Cyclic AMP and Cell Behavior in Cultured Cells

Cyclic AMP and Cell Behavior in Cultured Cells MARK C. WILLINGHAM Lnhoratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland

I. Introduction

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11. Effects on Morphology

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IV.

V.

V1.

VII.

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A. Fibroblastic and Embryonic Cells . B. Cells ofNeurona1 Origin . . . . C. Other Cells . . . . . . Growth Control and Contact Inhibition . A. Fibroblastic and Embryonic Cells . . B. Cells of Neuronal Origin . . . . C. Epidermal Cells . . . . . D. Miscellaneous Cells . . . . . E. Effects on the Cell Cycle . . . . Effects of CAMP on Biochemical Functions . A. Membrme Transport . . . . B. Enzyme Induction by CAMP . . . C. Production of Cell Products and Other Biochemical Functions . . . . . . . . . . Properties Mediated through the Cell Surface . . . A. Motility and Migration . . . . . . . B. Agglutination by Plant Lectins . . . . . C. Adhesiveness and Other Cell Surface Properties . . Malignancy and Differentiation . . . . , . A. Malignant Transformation in Cultured Cells and Tumorigenicity . . . . . . . . . . B. Differentiation in Culture . . . . . . . Concluding Remarks . . . . . . . . References . . . . . . . . . .

319 322 323 330 332 333 333 34 1 342 343 344 345 345 347 348 349 349 35 1 352 353 353 355 356 357

I. Introduction Adenosine 3‘,5‘-monophosphate (cyclic AMP, CAMP) has been accepted as a ubiquitous, central regulator of many biological processes (Robison et d.,1968; Pastan and Perlman, 1971). Its role in mammalian cells was originally delineated in the actions of extracellular hormones (Rall et d.,1971).cAMP was shown to be the intracellular “second messenger” for hormone-mediated cellular regulation. Many hormones act on their endocrine target tissues through activation of plasma membrane adenylate cyclase, elevation of intracellular cAMP levels, and tissue-specific reactions to this high cAMP level. Cytoplasmic CAMP-dependent protein kinases have been identified and offer one explanation for the mechanism of action of 319

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CAMP in these tissues. For example, liver cell plasma membranes respond to glucagon through activation of their adenylate cyclase, causing elevation of intracellular CAMP, and finally resulting in increased breakdown of the glycogen stores within the liver parenchymal cell (glycogenolysis). Thus glucagon’s action on liver cells, glycogenolysis, has been shown to be mediated through CAMP. In addition to its hormone-mediation functions, CAMP has been implicated in the regulation of many cellular processes, and it is the function of this article to summarize the wealth of information that has accumulated about the role of this nucleotide in cells in tissue culture. The use of tissue culture has allowed the study of the control of multiple cell functions. These spontaneously growing cells apparently have not repressed genetic information for many functions that are repressed in mature, differentiated cells. The need for growth control by CAMP is perhaps of little importance to a completely differentiated cell which may have stopped growing because of suppression of the genes responsible for spontaneous growth. However, interest in this control suddenly increases when these hypothetical genes express themselves and result in the spontaneous, uncontrolled growth manifested as malignancy. This is the rationale for studying the control of spontaneously growing embryonic or “fibroblastic” cultured cells and attempting to understand the transformation of their properties to those characteristic of malignancy in culture. The fact that this transformation often results in the ability of these cells to create transplanted malignant tumors in animals lends validity to this approach. We restrict this article to the functions of CAMP in cultured cells, deviating from this only to examine cells derived from culture (e.g., transplantable tumor cells), or cells in which the control mechanisms shown in culture can be shown in intact animals (e.g., epidermal cells). We also deal peripherally with the inescapable parallels that exist between embryonic cells in culture and embryogenesis or differentiation in vivo. The term “normal” cultured cells is used here to refer to the properties of many cultured cell types that are altered after malignant transformation. Among these “normal” properties are slow logarithmic growth rate, contact or density-dependent inhibition of growth, contact inhibition of movement, flat or spindly shape, high adhesiveness to substrate, poor agglutinability with plant lectins, low rate of membrane transport of nutrients, and inability to produce malignant tumors when transplanted into animals. To understand many of the agents or approaches used in these

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experiments, some understanding of cAMP and its metabolism is necessary. CAMP is produced intracellularly through the action of the membrane-bound enzyme adenylate cyclase. This enzyme catalyzes the conversion of adenosine triphosphate (ATP) to CAMP. CAMP can then be degraded b y a second enzyme (or enzymes), phosphodiesterase(s) (PDE), to yield the noncyclic derivative 5'adenosine monophosphate (5'-AMP). Thus the level of cAMP in cells can be regulated through the activities of these two enzymes, or its loss from the soluble cytoplasm through external excretion or binding by specific CAMP-binding proteins in the cell. When CAMP is measured in cells, it is the total intracellular CAMP that is measured. CAMP in the culture medium must be measured by different methods, and in most studies is not measured at all. One can artifically alter intracellular CAMP levels by several means. One way is to add CAMP itself to the medium in which the cells grow. Unfortunately, CAMP itself is poorly diffusible through the cell membrane. As a result, more permeable analogs are often used, such as N Z , 02-dibutyryl CAMP (Bt,cAMP), N'-monobutyryl CAMP (BtcAMP), and the 8-bronio derivative of cAMP (8BrcAMP). These agents possibly act through their similarity to CAMP, presumably binding to the same intracellular receptor to which cAMP binds. Another way to increase CAMP in cells is to stimulate adenylate cyclase activity. Nature has provided us with many hormones that can do this in hormonally responsive cells (epinephrine, ACTH, glucagon, and others) but, often, cells in culture may not be hormonally responsive. Another class of compounds that naturally occur are the prostaglandins, some of which activate adenylate cyclase in cultured cells, one ofthe most effective being prostaglandin El (PGE,). An additional method of raising CAMP is through inhibition of its degradative enzyme PDE. Many drugs have this property, including xanthines (aminophylline, theophylline, 1-methyl 3isobutyl xanthine), papaverine, and other synthetic inhibitors (e.g., Roche compound no. 1724). The maintenance of growth in cultured cells often requires serum to b e included in the medium, particularly its macromolecular constituents. Deprivation of serum factors results in the elevation of CAMP levels in many cell types in culture, so this is another method of raising CAMP, although it certainly affects many other processes. Lowering CAMP is considerably more difficult. The addition of fresh serum factors causes a fall in cAMP in some cells. Treatment of cells with proteases, such as trypsin, results in lowered cAMP levels. Insulin, which acts in a manner opposite glucagon in liver cells,

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causes lowered cAMP levels by inhibiting adenylate cyclase, activating cAMP PDE, or both. One can see from these agents that agents that lower cAMP are often growth stimulators in culture, a fact consistent with the inhibitory effects of cAMP on cell growth. We should not ignore the evidence regarding another cyclic nucleotide, guanosine 3’,5’-monophosphate (cGMP), in cell behavior. Studies have appeared which demonstrate changes in cGMP during various growth or differentiated responses, particularly in lymphocytic cells. These changes are opposite those seen in CAMP. This has led to the concept of a balance between these nucleotides (Goldberg et al., 1974), which regards the cAMP/cGMP ratio as the regulatory determinant in cell function, rather than just the actual level of one nucleotide or the other. This concept has remained unsupported in the cultured cells we consider in this review, since inclusion of cGMP or its analogs in these systems has consistently failed to show the type of regulation one might expect if it truly regulates any functions on its own. In differentiated cells, such as polymorphonuclear leukocytes, cGMP has been shown to be effective in regulating specific functions, and these cases are mentioned under their particular property (such as migration for PMNs; Section V,A). The literature search for this review was conducted utilizing the MEDLINE retrieval system (National Library of Medicine).

11. Effects on Morphology

The study of CAMP’Soverall effects on cell morphology has been generally limited to in vitro tissue culture systems. The majority of these observations have been at the light microscope level and involve gross changes in cell shape. Cells growing in dishes, flasks, or suspension culture usually undergo relatively few changes in gross morphological appearance. Cells that have a predominantly round shape can, with various treatments, become attached to a substrate, protrude blebs or microvilli, emit spindly processes, or completely flatten against the underlying surface. Cells crowded by other cells can become rounder, and flatten to a limited degree and become box-shaped, or produce long, stretched-out processes and become spindly. Conversely, cells that are predominantly flat or spindly can become round, detach themselves from their substrate, and float away, or change the appearance of their surfaces by protruding blebs or microvilli. Accompanying these gross changes can be many less obvious intracellular events involving alterations in organelles, membranous structure, or other subcellular components.

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Change in shape also may be accompanied by or closely related to changes in motility or adhesiveness (discussed in Section V,A,C), properties that may be intimately involved in many of the morphological alterations we observe. Since cAMP has been implicated as a normal regulator of many cell functions, it is not surprising that alterations in its metabolism have profound effects on cell morphology. Since cell morphology is one of the more easily observed cell functions, it is also not surprising that this was one of the first areas in which cAMP was found to be a regulator of cell behavior.

A.

FIBROBLASTIC AND EMBRYONIC CELLS

It had been known for some time that “normal” cultured cells derived from either mature or embryonic tissues often underwent morphological changes with malignant transformation. This inspired many investigators to use the morphology of cells as a tool in studying the mechanism by which cells were transformed. With the knowledge that cAMP was an important mediator of hormonally induced changes in function in target tissues, and that it had been shown to be an ubiquitous compound even regulating metabolic events in bacteria (Pastan, 1972), investigators tried to find a regulatory function for cAMP in malignant transformation. The obvious first place to look was the most easily observed parameter, morphology * In 1971, six separate articles were published dealing with the effects of cAMP on morphology in transformed and normal cells. From one laboratory, three reports appeared (Johnson et al., 1971a,b; Johnson and Pastan, 1971) showing that treatment of many cell types [rat sarcoma cells induced by ROLLSsarcoma virus (RSV), XC; L929; RSV hamster; human osteosarcoma; polyoma virus (Py) mouse cells; uncloned mouse embryo fibroblasts, MEFI with CAMP, or more frequently its butyryl derivatives (BtcAMP, BtcAMP), or agents which elevated intracellular cAMP levels (prostaglandins, xanthines), changed cell morphology significantly. Specifically, Bt,cAMP caused most trunsforrned cells in culture to look more like their normal parent. They either became flatter or more spindly, rather than round (Fig. 1). Normal cells at light density became flatter and more spindly than usual (Fig. 2). This phenonemon was shown to be readily reversible. From another laboratory it was reported (Hsie and Puck, 1971; Hsie et al., 1971) that cells derived from Chinese hamster ovary (CHO) became elongated and grew in parallel arrays with BhcAMP

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FIG. 1. L929 cells grown on glass cover slips at 37°C in medium containing 10% cell serum with (B) or without (A) 1 mh4 Bt,cAMP for 24 hours. Phase-contrast. x410.

treatment. This growth pattern was reversible, and was enhanced by the addition of testosterone (perhaps further elevating intracellular CAMPlevels). Surface blebbing activity, called “knobs”, disappeared with this treatment. These elongation changes were inhibited by agents that interferred with microtubular function (Hsie and Puck, 1971; Johnson et al., 1971b).

FIG.2. 3T3-4 cells grown on plastic dishes at 37°C in medium containing 10% calf serum with (B) or without (A) 1 mM Bt,cAMP for 24 hours. Phase-contrast. X 130.

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Another article (Sheppard, 1971) showed that BtcAMP caused a more spindly morphology in transformed cells. The incubation of normal cells (contact-inhibited) at high density with BtcAMP caused little change in cell shape, a fact later found to show that, when contact-inhibited cells are closely packed together, their ability to flatten is inhibited because of crowding. These same cells treated at light density, however, showed marked flattening (Johnson and Pastan, 1972a) (see Fig. 2). Subsequently, Johnson and Pastan (19724 showed morphological effects of Bt,cAMP on other normal and transformed cells (Swiss and Balb-3T3, Py-3T3, MuLV-3T3, MSV-3T3, SV40-3T3, L-2071, BHK-21), all characterized as either flattening or developing a spindly shape. This flattening in normal cells was also reported (Seifert and Paul, 1972) with increased CAMP levels when cells were grown in medium containing low concentrations of serum. The changes in CHO cells were filrther described (Puck et al., 1972), and the forniation of surface “knobs” (blebs) and their disappearance after Bt,cAMP treatment were discussed. Gazdar et al. (1972) described reversible niorphological elongation and flattening in 3T3 and muriiie sarcoma virus (MSV)-transformed 3T3 cells with Bt,cAMP treatment. Otten et al. (19724 showed that a morphological criterion of transformation (vacuolization) in another cell system (chick embryo cells infected with a virus temperature-sensitive for transformation) could be retarded by BtzcAMP treatment, and that transformation in these cells was in fact accompanied by a fall in CAMP levels. Reviews discussing many of these articles have appeared (Pastan et al., 1974; Pastan and Johnson, 1974). Other agents have been shown to produce morphological changes of the same general type. Phenethyl alcohol (Wright et al., 1973) and sodium butyrate (Wright, 1973) produce elongation of CHO cells, but the effect of these agents on CAMP metabolism was not studied. In fact, sodium butyrate has been reported to raise intracellular CAMP levels in another system (Prasad et al., 1973b). Johnson et al. (1974) showed that N6-substituted derivatives of adenine can produce some of the morphological responses attributed to CAMP, without actually affecting CAMP levels in the cell. The possibility exists that these N6substituted compounds, most of which do not exist normally in a free state in cells, may mimic the action of CAMP in some way. These studies mainly point out that morphological changes of this type may not necessarily be due to alterations in CAMP itself in cells, but that some compounds may act through the same effector mechanisms that CAMP regulates under more normal conditions. Other alterations in

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media conditions, of course, affect morphology, sometimes in a manner similar to CAMP effects. Paul (1973) showed that low concentrations of leucine caused spindly morphology in SV40-3T3 cells. Since CAMP was not measured in these cells, the question remains whether leucine deprivation causes increased CAMP levels, or whether morphological changes with high cAMP levels are due to decreased leucine uptake. Curtis et al. (1973) noted a change in cell volume with changing cAMP levels. Storrie (1973) showed that cell rounding due to EDTA or concanavalin A (Con A) treatment of CHO cells could be slowed by treatment with BtcAMP and testosterone. He later showed (Storrie, 1974) that the effects of Con A on these cells was diminished by decreased temperature or inhibition of cell respiration. Further, the binding of Con A to these cells was unchanged by Bt,cAMP treatment. One might conclude from these studies that CAMP can regulate adhesiveness and morphology in such a way that other treatments that cause changes in these functions can be overridden. Patterson and Waldren (1973) showed, as had been implied earlier (Hsie and Puck, 1971; Johnson and Pastan, 1971), that morphological changes with BtcAMP are independent of new RNA and protein synthesis. That CAMP’Seffects are cytoplasmic in character was further demonstrated by Schroder and Hsie (1973), who showed that enucleated cells could be made to elongate by agents that cause increased cAMP levels in whole cells (BtcAMP, PGEJ, and that this action was prevented by inhibitors of microtubular function (vinblastine). De Asua et al. (1973) demonstrated the effects of BtcAMP and theophylline on the growth of baby hamster kidney (BHK) cells in agar, as well as on normal substrates. BHK-21 cells grew in agar when they were stimulated with exogenous insulin, perhaps partly as the result of a fall in intracellular CAMP levels following insulin treatment. This stimulation with insulin was inhibited by BbcAMP. In addition, insulin imparted a transformationlike morphology to these cells when anchored to substrates, a reaction prevented by BtcAMP. Carchman et al. (1974) showed a similar reversion to normal morphology with BtcAMP treatment in normal rat kidney cells transformed by Kirsten MSV. This reversion was unaffected by derivatives of cGMP. Mitchell et al. (1973) and Korinek et al. (1973) reported spindly fibroblastlike morphology of XC cells after BtcAMP treatment. It had been known that treatment with agents that lower CAMP levels dramatically (serum, trypsin and other proteases, insulin) was sometimes accompanied by morphological changes opposite those

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caused by increasing CAMP. The major question that arose about many of these studies of morphological changes with agents that elevate or lower CAMP levels was whether these effects on cAMP were simply coincidental occurrences and whether the actual morphological change might be related in some way to the exogenous agent's "toxicity." To answer this question, Willingham et ul. (1973) isolated a temperature-sensitive mutant cell from nonmutagenized cultures of Swiss 3T3 mouse cells. This variant cell type could be cloned and propagated with consistent properties. Unlike most mutants with temperature sensitivity, this cell was sensitive only to changes in temperature rather than a specific range of temperature. As a constant growth temperature (23"-39"C), it behaved exactly like a normal 3T3 cell; it was contact-inhibited for growth, had the same morphology and growth rate, and had the same responses to exogenous agents that raise CAMP levels. With a sudden change in temperature, however, the intracellular cAMP levels in this cell fell to very low levels within seconds, followed by a loss of adhesiveness to the substrate (the property by which it was originally derived) and, within 10-15 minutes, b y an extreme change in morphology from flat to completely round with a retraction of processes (Fig. 3).

FIG.3. 3T3 cAMP"'1 cells grown at 37°C in medium containing 10% calf serum before (A) and 10 minutes after (B) changing the culture temperature to 23°C. (See Willingham et ol., 1973.) Phase-contrast. x 130.

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Again the question whether this sudden change in cAMP levels followed by morphological change was coincidental arose, but it was shown that a variety of agents that prevented the initial fall in cAMP levels (by raising CAMP) could completely abolish both the change in adhesiveness and the later severe alteration in morphology. Further, the characteristics of this cell were such that, within 30-60 minutes, cAMP levels began to return to normal, with a large overshoot in cAMP levels in 90-120 minutes, attaining a constant normal level in 3-4 hours. During this overshoot phase the cells were refractory to further changes in temperature, showing no sudden morphological change with changes in temperature. This article contains strong evidence that cAMP has a physiological role in controlling the morphology of cells. Studies have been undertaken to relate gross morphological changes to subcellular organelle function (Porter et al., 1974; Willingham and Pastan, 1975a). It had been pointed out that agents that interfere with microtubular function prevent most of the gross morphological changes due to elevated cAMP levels (Hsie and Puck, 1971; Puck et al., 1972; Schroder and Hsie, 1973; Johnson et al., 1971b). It is not surprising, then, that Bt,cAMP treatment results in redistributed or increased microtubular structures (Porter et aZ., 1974; Willingham and Pastan, 1975a). In addition, the subcellular distribution of microfilaments is altered (Willingham and Pastan, 1975a) (Fig. 4). Other occasions in which cell morphological changes have been studied indicate a corresponding alteration in these structures with gross morphological change (Porter et al., 1973; McNutt et aZ., 1973). From this type of study, the concept has emerged that microtubules form a structural or “skeletal” unit of gross morphological structure, whereas microfilaments are active, dynamic elements which position portions of the cell during morphological change. Supporting this further is the evidence that microfilaments contain contractile protein components, mainly actin (Abercrombie et aZ., 1973). A model by which cAMP might regulate microtubular and microfilamentous structures, and thus cell shape and motility, has been proposed and is shown in Fig. 5 (Willingham and Pastan, 1975a). The means by which contractile proteins actually move portions of the cell has remained obscure, since the other factor in contractile protein interactions, myosin, has remained elusive in its cellular location. Recently, Willingham et al. (1974) showed that a substance antigenically indistinguishable from cellular myosin is present on the exterior of the cell membrane. They suggest that only the tail end of

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NORMAL

CELL TREATED WITH Bt2cAMP

r-

FIG.4. A diagrammatic representation of the morphological changes in L929 and 3T3 cells after Bt,cAMP treatment.

myosin might be exposed on the outside surface of the plasma mernbrane, with the remainder of the molecule protruding through to the inside. This would place its HMM-enzymic and actin-reactive end on the inner surface of the plasma membrane, available to react with microfilamentous actin. Perhaps an interaction of these two molecules would cause myosin, anchored in the plasma membrane, to slide along the actin of the microfilaments, loosely anchored in the

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MARK C. WILLINGHAM microtubule assembly (process extension)

Cyclic AMP

t increases

substratum adhesiveness

inhibit

tmicrofilament-mediated contraction (process retraction) FIG.5. A proposed model of the mechanisms by which CAMPregulates cell shape and motility. (See Willingham and Pastan, 1975a.)

cytoplasm, and thus move the surface membrane with respect to the cytoplasm. This type of mechanism of cell motility has been proposed before (Abercrombie et d.,1973). Process extension could therefore involve membrane-bound myosin reacting with cytoplasmic microfilamentous actin. Process retraction, however, might involve myosin in an intracytoplasmic location reacting with microfilament arrays extending along the cell process. Further studies are needed to confirm this hypothesis, but it lends mechanistic logic to the manner by which cells move and correspondingly change shape. Since CAMP’Sactions involve cell motion (discussed in Section V,A) and change in cell shape, it is likely that this regulatory nucleotide might also control the interaction and subsequent mechanical actions of these contractile proteins. How this regulation takes place is still unknown. B. CELLS OF NEURONALORIGIN In a manner similar to the study of fibroblastic cells, considerable interest in the effects of CAMP on neuronal cells centered on morphological changes. An added feature of some of these cells, however, was the relationship of morphological change to differentiation. A proposed criterion of differentiation found in some of these cells, particularly those derived from neuroblastoma, is the irreversibility of cell process extension without cell death. This subject is discussed in more detail in Section V1,B. Mouse neuroblastoma cells were shown to undergo axonal formation with BbcAMP treatment (Prasad and Hsie, 1971). This was seen with 1 mM BtcAMP in 24 hours, being maximal in 3-5 days. After 3 days the changes appeared irreversible. C1300 mouse neuroblastoma

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showed similar changes with neurite extension, but the effects of BtcAMP were said to be reversible after 24 hours of treatment (Furmanski et al., 1971). Prasad (1972) also showed that PGE, was effective in producing the same effects. It had been previously shown that serum deprivation of neuroblastoma cells results in process extension (Seeds et d., 1970). The change with BbcAMP was later shown to be absent with S ’ A M P or native CAMP, but present with N BtcAMP (Waymire et al., 1972). Similarly, these cells were unaffected by ATP, ADP, or cGMP (Prasad, 1972). Prasad et al. (197313) measured CAMP levels in response to numerous agents that induce differentiated responses. Even though they induced neurite formation irreversibly, x-irradiation or Gthioguanine did not alter CAMP. PGEI, a synthetic PDE inhibitor (Roche no. 1724), serum-free medium, and BUdR all increased CAMP levels and caused neurite formation. Curiously, buytric acid raised levels but, through some other interfering action, did not produce neurite formation. Vinblastine and cytochalasin also interfere with neurite formation. The morphological changes were shown to require new protein synthesis in these cells (Prasad et al., 1972; Furmanski et al., 1971). In a glial tumor cell line (C-6), Schwartz et al. (1973) showed that Bt,cAMP produced morphological changes similar to those produced by bromodeoxyuridine (BudR). These were mainly cell flattening and elongation, particularly at confluency. Edstrom et al. (1974) showed the effects of BbcAMP and PGEt on human glioma cells in culture. Again, this morphological change involved the formation of long, thin processes, but was reversible. MacIntyre et al. (1972) reported that human astrocytoma cells show reversible morphological change with BtcAMP treatment. However, human neuroblasts showed an irreversible morphological change with Bt,cAMP characterized by many extensions and extracellular microfilamentous mats. Roisen et al. (1972a,b) reported that CAMP and BbcAMP both produced axonal maturation in explants of sensory ganglia. This was seen as increases in both the number and length of axonal extensions. Fetal sensory ganglia have been shown to produce more neurite outgrowth with BtcAMP treatment (Haas et al., 1972), a response requiring new protein synthesis. Shapiro ( 1973) reported the appearance of multiple cell processes after N-BtcAMP treatment in fetal rat brain cultures. As can be seen, all these morphological changes involved the production of cellular processes, a result thought to represent a beginning response in the direction of differentiation for neuronally derived cells.

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C. OTHERCELLS

1. Melanoma Cells Johnson and Pastan (1972b) showed increased pigment production with BtcAMP treatment of mouse melanoma cells, mostly an irreversible change. Kreider et al. (1973) reported that B16 melanoma cells produced more pigment in response to CAMP, BtcAMP, caffeine, or theophylline. This was accompanied b y cellular hypertrophy and exaggerated dendrite formation with increasing cell volume. O’Keefe and Cuatrecasas (1974) showed increased size and pigment production of melanoma cells after the addition of cholera toxin (thought to increase CAMP through activation of adenylate cyclase). 2. Muscle Cells Wahrmann et al. (1973) showed that BtzcAMP produced elongated narrow processes in myoblasts of cell line L,D, but that this was not accompanied by myotube formation, an index of differentiation. In fact, BbcAMP was shown actually to inhibit myotube formation. Reporter and Norris (1973) showed that CAMP induced long, thin processes in primary rat muscle cells. Primary chick myoblasts were also inhibited in fusion by BtzcAMP (Zalin, 1972,1973). Colony formation of human rhabdomyosarcoma cells in agar was inhibited by Bt,cAMP treatment (Sandor, 1973). In normal culture these same cells produced fiberlike extensions and showed central rounding with condensed nuclei after exposure to Bt,cAMP.

3. Adrenal Cells Adrenal cells have been reported to show a morphological response to CAMP different from that of other cells. They generally become round with increased CAMP levels. A line of adrenal tumor cells that respond to ACTH showed rounding after ACTH treatment (thought to increase CAMP levels) (Yasumura et al., 1966). Masui and Garren (1971) showed that these same cells showed morphological change with CAMP treatment similar to that caused by ACTH. O’Hare and Neville (1973a) grew adrenal cells derived from a normal adult rat and showed cellular retraction and rounding with cAMP or ACTH treatment. Milner (1972) showed that primary fetal rat adrenal cells respond morphologically to cAMP in some of the ways they respond to ACTH, but not all. Most of these were small ultrastructural changes.

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4. Miscellaneous Cells Werthamer et al. (1974) has reported that some human lymphocytic tumor cells become fibroblastlike after Bt,cAMP treatment. Seller and Benson (1973a,b) showed that Erhlich ascites tumor cells i n uiuo increased in size with cAMP treatment. Naseem and Hollander (1973a,b) reported that mouse myeloma MPC-11 cells become large and vacuolated after cAMP treatment. Nose and Katsuta (1974) reported extensive cytoplasmic elongation with Bt,cAMP treatment of cultured rat liver cells.

111. Growth Control and Contact Inhibition

A.

FIBROBLASTIC AND EMBRYONICCELLS

Of CAMP’Smultiple effects on cells in culture, growth control is perhaps the most significant and the most interesting. Unlike most highly differentiated cells, cells in culture have been generally selected for their ability to grow spontaneously. As a result, overall regulation of cellular function is commonly manifested in their growth properties. Highly differentiated, hormonally responsive cells use cAMP to turn on their differentiated cell functions, usually the production of some cell product or metabolic activity. Rarely are these cells called on to proliferate and increase their number. For example, adrenal cells respond to ACTH by activating adenylate cyclase in their plasma membrane, thus increasing intracellular CAMP, and this in turn increases steroid production. The liver cell, as another example, responds to glucagon by raising its cAMP levels through this same activation of membrane adenylate cyclase, and this in turn increases glycogenolysis intracellularly. In neither case does cAMP usually act to induce cell replication. From this one might surmise that cAMP is a positive regulator of di.fferentiated cell function. In cells that are normally turned on for growth regulation, however, CAMPappears to act in another manner. Examples of such cells are the numerous continuous untransformed cell lines, many of which are embryonic cells and all of which are permanently turned on for continuous growth in low-density culture. In these cells cAMP can show itself as an effective negative regulator of cell multiplication. Unfortunately, all growth regulation is much more complicated and, in some cells, such as lymphocytes, the call for differentiated function that also involves increased cell multiplication appears to involve CAMP in both negative and positive regulatory roles.

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Our discussion in this section is limited to the regulatory role of cAMP in spontaneously growing cells in culture. In almost every instance we discuss, evidence has accumulated that clearly shows CAMP’Sability to slow down or stop cell replication in cultured cells. The interest in this regulation is all the more important in light of the study of transformed derivatives of these cells and their deficiencies in the cAMP regulatory chain. Even though most of these “fibroblastic”, cultured, spontaneously growing cells show autonomous replication with the proper culture conditions, many do not show this spontaneous growth ability when transferred to an in vivo host. This property has lent these cells a cloak of normality in that they are not tumorigenic cells. Derivatives of these cells transformed by a variety of agents are sometimes quite tumorigenic and fatal to their host. When these tumor-producing cells are transferred to tissue culture, they show several properties in culture that are different from those of their nontumorigenic parents. As a result, normal and malignant cells have been characterized in culture, and a list of transformed phenotypic properties has been compiled. At the head of this list is growth; normal cells are said generally to grow slower than their transformed, malignant offspring and to show a tendency to stop growing when they reach a high cell density in culture. This density-dependent inhibition of growth is absent in most transformed derivatives in which the cells continue to replicate until they either exhaust the culture medium of nutrients (“killing” the in vitro host), or detach from their underlying substrate, either surviving or dying in suspension. A subdivision of density-dependent inhibition of growth is a condition in which cell replication ceases as the available surface area of the substrate is used up, with little or no overlapping of cells. This is called, by many, contact inhibition of growth, being derived from the phenomenon of inhibition of cell overlaps on a substrate during cell movement (contact inhibition of movement). The phenomena of contact and density-dependent inhibition of growth are separable phenomena (at least in our opinion), and only a few normal cell lines show true contact inhibition of growth, whereas almost all show density-dependent inhibition of growth. Without careful observation and morphological criteria, these phenomena are easily confused. I n addition, from time to time, one particular cell line may begin to lose contact inhibition of growth, usually by selective overgrowth of a non-contact-inhibited subpopulation. Multiple stored frozen samples of the same cell line may, after thawing, show variability in these properties. It is relatively important, then, for inves-

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tigators interested in contact inhibition to monitor their cell lines constantly for loss of this property, which can generally be corrected by recloning and selection of a resultant clone that preserves this phenotype. This problem introduces a difficulty in interpretation of results in various publications, unless the specific property of the cell line has been monitored and stipulated in each article or in previous publications from each laboratory. The role of CAMP in growth control has been delineated in numerous publications in the last few years. Controversies have arisen among various laboratories regarding conditions of culture or biochemical measurement. What we attempt to do is to stress what we consider the most likely and most well-supported evidence, but all the evidence available is presented. Historically, the first two publications dealing with CAMP and growth control appeared in 1968, when Ryan and Heidrick (1968)reported that CAMP itself inhibited the growth of HeLa and L cells in culture. In addition, they reported that BtcAMP also showed some inhibitory effects. In the same year, Burk (1968) showed that CAMP decreased the growth of BHK, Py-transformed BHK, and (Bryan) RSV-transformed BHK cells. Caffeine and theophylline also inhibited the growth of these cells. He also noted that the more rapidly growing Py transformant had lower adenylate cyclase activity, implying that, in the transformed cell, the resulting lower CAMP levels from this unilateral event might be consistent with a more rapid growth rate. Heidrick and Ryan (1970) fiirther showed that cAMP inhibited the growth of FL ammion cells and human epidermoid laryngeal carcinoma (HEp-2) cells, and slightly inhibited that of human diploid embryonic lung fibroblasts (WI-38). They later observed increases in CAMP with increasing cell density in transformed L cells (Heidrick and Ryan, 1971), perhaps indicating that CAMP levels increased with depletion of serum growth factors from the medium. They stated that highly tumorigenic L cells showed lower cAMP levels than less tumorigenic cells. Hsie and Puck (1971) showed that CHO cells are inhibited by treatment with BtcAMP at lop3M . This effect on growth requires higher concentrations than that required to show the earliest morphological changes (0.3 x M ) . Sheppard (1971) demonstrated reversible growth inhibition of transformed cells (Py-3T3), but his claim of “restoration” of contact inhibition of growth was later found to represent medium depletion (Johnson and Pastan, 1972a; Smets, 1972; Paul, 1972; Grimes and Schroeder, 1973; Rozengurt and Pardee, 1972). Further, the lack of effect on normal 3T3 cells (Shep-

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pard, 1971) probably represented too high an initial plating density, since these cells are quite sensitive to growth inhibition by Bt,cAMP (Willingham et al., 1972; Johnson and Pastan, 1972a). Johnson and Pastan (1971, 1972a) demonstrated the effects of Bt,cAMP (plus theophylline) on normal 3T3 cells (Balb and Swiss) and on several transformed cells (SV40-3T3, Py-3T3, MuLV-3T3, MSV-3T3, L-2071). This treatment was shown to decrease the growth rate of normal and transformed cells, but not to restore contactinhibited growth to transformed cells. Further, normal cells, in addition to showing slower growth rates, also showed a lowered saturation density, most easily explained by noting the extremely flat morphology of these cells and thus their earlier cell-to-cell contact. Sniets (1972) showed that Bt,cAMP slowed growth in 3T3 cells transformed by SV40. Otten et nl. (1971) measured CAMP levels in normal and transformed cells and found that CAMP correlated inversely with growth rate; that is, transformed cells with low CAMP levels grew faster, whereas normal cells with higher CAMP grew slower. In addition, they showed that as 3T3 (contact-inhibited) cells approached confluency and began to touch, CAMP levels began to rise dramatically, reaching a maximum at confluency, with subsequent growth arrest in the GI phase of the cell cycle (Otten et nZ,, 1972b). Willingham et (11. (1972) showed that cells in this condition, when trypsinized and replanted, reinitiated growth, beginning DNA synthesis in 12-16 hours. This reinitiation from G, arrest was prevented, however, b y raising CAMP levels at replanting b y incubating in Bt,cAMP. Further, raising CAMP after a critical 3- to 4 h o u r period after planting seemed to make no difference in subsequent DNA synthesis, so this period of sensitivity to high CAMP levels represented a cell cycle block in early G,. They also showed that CAMP could block the cell cycle at a point in G, and prevent cells from entering mitosis even though they had completed DNA synthesis. Chronic treatment of 3T3 cells at light density with BtcAMP induces synchrony in that many of these cells became arrested in GI (K. Olden, personal communication). Similar findings regarding CAMP’Sspecific control of the cell cycle have been reported for other cells. Froehlich and Rachmeler (1972, 1974) showed quite clearly that, in human diploid fibroblasts, entry into the cell cycle from GI arrest at confluency requires a fall in CAMP. By trypsinization and replanting or by addition of fresh serum, they showed a delay in the onset of DNA synthesis with BbcAMP treatment. Unlike 3T3 cells, which lose their ability to be

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blocked in 3-4 hours, their cells retain this control up to 8 hours after stimulation. Difference in specifics of control (required concentrations of inhibitors or length of lag or control point time) are probably due to differences in cell types among these systems. Burger (Bombik and Burger, 1973; Burger et al., 1972a,b) demonstrated another system of stimulation of growth from confluencyarrested cells. Pronase treatment of 3T3 cells for short periods was reported to produce synchonous DNA synthesis, a response blocked by Bt,cAMP treatment near the time of enzymic treatment (k30 minutes). The stimulation of growth following insulin or new serum addition was similarly prevented by Bt,cAMP (Bombik and Burger, 1973).These effects were not seen with other nucleotides (cGMP or cCMP), or with buytric acid or 5'-AMP. By measuring CAMP levels during stimulation, Burger et al. (1972b) demonstrated a fall in CAMP initially followed b y a rise in S phase, and then a sudden drop in levels at the time of mitosis. They also found this drop to follow serum addition, trypsin, and other proteases that stimulate GIarrested confluent cells to grow. Otten et (11. (1972b) showed that CAMP rose at confluency in contact-inhibited cells, both 3T3 and human sinus polyp diploid fibroblasts, (MA308), and that treatment with fresh serum, trypsin, or insulin produced a fall in CAMP levels of considerable duration (hours). Further, they showed that the elevation of CAMP due to PGE, activation of adenylate cyclase was inhibited by the addition of insulin. Zacchello et n l . (1972)reported rising adenylate cyclase with increasing cell density in human fibroblasts in culture. Anderson et nl. ( 1 9 7 3 ~ showed ) increased CAMP at confluency in cloned normal rat kidney (NRK) cells, which are contact-inhibited, This increase was accompanied b y an increase in adenylate cyclase activity with cell density, b u t failure of a corresponding increase in PDE activity. The divergence of these enzymes began near the point of significant cell contact when CAMP levels were beginning to rise. All these articles tend to leave convincing evidence that (1)CAMP levels are high at confluency in contact-inhibitied cells, (2) growth stimulation of these cells from this state requires a fall in CAMP levels, and (3) prevention of this fall in CAMP prevents the subsequent stimulation of growth. From this evidence the postulate is well supported that high CAMP at confluency in contact-inhibited cells is responsible for their cessation of growth and arrest in G,. Sheppard (1972) measured CAMP levels in 3T3 cells at varying densities (but always above 10' cells/cm2)and came to the conclusion that CAMP levels were high at all times, even at low density. He also

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reported that the levels of cAMP in rapidly growing transformed cells were lower than in slower growing normal cells. Subsequently, he published more evidence (Bannai and Sheppard, 1974) in which he reports a rise in cAMP as 3T3 cells approach confluency. The new data show a precipitous rise in cAMP at 3 x 103 cells/cm2, a point still somewhat short of confluency or even extensive cell-to-cell contact in our laboratory. One problem is that the comparison of densities is difficult, since light-density cells were grown in roller cultures, rather than in flasks as the high-density cells were. First, in this type of culture it is difficult to assess density accurately, and almost impossible to evaluate morphology as a control for proper culture conditions. Second, and probably more important, the cells were assayed only 24 hours after being planted (and presumably trypsinized). Trypsin treatment and replanting of 3T3 cells produces a synchronous cell population, a synchrony that lasts at least 30 hours (at the time of mitosis) (Willingham et aZ., 1972). Therefore cAMP measured at this time probably represents the levels in cells at late S phase of the cell cycle. Second, trypsin treatment results in an extremely flat morphology during the first cell cycle following replanting (Willingham and Pastan, 1975a), perhaps indicative of high cAMP levels and, for this reason, others working in this field (Otten et al., 197213) routinely wait at least beyond the end of the first cell cycle after planting to assay CAMP. Further evidence for the high cAMP levels within 1 day after trypsinization is provided by the observation that the 3T3 cAMPtcsmutant (Willingham et al., 1973)is relatively refractory to morphological change (caused b y falling CAMP levels) during the first day after trypsinization (Willingham and Pastan, 1975a). It has been appreciated by many that trypsin treatment is of sufficient severity to cells that they recover slowly, probably not completely until the next mitotic event (see also Russell and Pastan, 1973). Other studies using similar techniques and yielding results similar to those in Sheppard's earlier article (Oey et al., 1974; Burstin et al., 1974) perhaps suffer from some of these same problems, if not from the fact that no points were given below cell densities of 104 cells/cm2 in either case. Grimm and Frank (1972) showed that in embryonic rat cells (which stop growing in serum-deprived medium in GI) cAMP rises with serum deprivation, and PDE activity is increased with the readdition of serum, presumably lowering cAMP levels to allow resumption of growth. Frank (1972) also showed that BbcAMP reversibly suppresses thymidine incorporation into these cells, similar to serum deprivation, and that subsequent stimulation of serum-deprived cells

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with fresh serum is blocked by BbcAMP. Pardee (1974) demonstrated that multiple treatments that suppress growth somewhere in GI (serum starvation, BbcAMP, isoleucine deprivation, glutamine deprivation, papaverine) seem to block the cell cycle at a single point in G, (a restriction point, or R point) in BHK cells. A transformed derivative of this cell (Py-BHK) seemed to lack this same control point. Many studies have dealt with the effects of agents or treatments that raise cAMP levels on growth control in culture directly in both normal and transformed cells. The majority of these studies that follow show that cAMP is a negative regulator of cell growth. Paul (1972) showed that BbcAMP slows the growth of SV40transformed 3T3 cells, even though the cells continue to grow slowly. Remington and Klevecz (1973) treated CHO cells with BbcAMP and found that many were arrested in Gz rather than in GI, although they still found cells in G, and even some in S. D’Armiento et al. (1973) showed that the slowing of growth due to low p H of the culture medium was accompanied by a rise in cAMP in WI-38 cells. Transformed SV40-3T3 cells continued to show low cAMP levels and continued to grow at either high (7.7) or low (6.6) pH. Smets (1973)demonstrated a shift in preponderance of cells in GI and S, to G, after BtcAMP treatment of human EB virus-transformed lymphocytes. A similar result was reported to have been found in SV40-3T3 cells. Carchinan et al. (1974) showed rising CAMP levels at confluency in NRK cells, but also showed that transformed NRK cells (KNRK) had low levels. A temperature-sensitive mutant of KNRK (temperaturesensitive for transformation) showed decreased cAMP levels at the transformed phenotypic temperature, where the cells showed faster growth and loss of contact inhibition. Bt,cAMP slowed the growth of KNRK cells, as well as the growth rate of normal NKR cells. BtzcGMP and 8BrcGMP had no effect. Rozengurt and Pardee (1972) reported that in CHO cells the inhibitory effects of BbcAMP on growth could be partially counteracted with increasing serum concentrations. Tee1 and Hall (1973) showed inhibition of growth in human nasopharyngeal carcinoma (KB) cells with Bt,cAMP treatment which was reversible and showed some synchronization of cells in GI. Kram et a2. (1973) showed that effects of serum deprivation on cellular transport (leucine, uridine) phenomena could be mimicked by PGE, or Bt,cAMP treatment. They showed that, with serum starvation, CAMP increases but returns to normal with the readdition of new serum. Bt,cAMP was shown to de-

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crease incorporation of t h ~ m i d i n e - ~ after H stimulation with fresh serum in Balb-3T3 cells. From this evidence they proposed that CAMP might be a regulator of the “pleiotypic” reactions of cells to growth conditions. Kram and Tomkins (1973) showed that cGMP antagonized these changes in transport due to CAMP if observed in serum-starved cells. Seifert and Paul (1972) demonstrated an increase in CAMP in 3T3 cells grown in low-serum medium, and an increase in CAMP at high density in both high- and low-serum media. The addition of fresh serum to these cells caused a rapid fall in CAMP levels. All these articles demonstrate the role of CAMP as a negative regulator of cell growth, and that other negative regulators (such as serum deprivation) may also function at least in part through CAMP. K ~ r t hand Bauer (1973) reported decreased growth of a RSVtransformed mouse cell line (D4) with BtcAMP and theophylline. Lower amounts of Bt2cAMP became more effective in inhibiting growth as cell density increased. Wright ( 1973) showed decreased growth of CHO cells with BbcAMP treatment, along with an inhibitory effect of phenethyl alcohol at a higher concentration. The effects of this latter agent on CAMP metabolism are unclear. Grimes and Schroeder (1973) showed decreased growth of Py-3T3 cells with Bt,cAMP treatment, but no restoration of contact inhibition. Blat et (11. (1973) reported a decrease in growth rate of BHK and Py-RHK cells in response to BtcAMP. BtcAMP reduced the eventual saturation density of BHK (normal) cells, but not of their transformed derivatives, in agreement with previous reports (Johnson and Pastan, 1972a). Aujard (1971) reported decreased growth of KB cells with CAMP treatment. D. B. Thomas et al. (1973) showed inhibition of growth in a murine mastocytoma cell line by BtcAMP, reporting a relative arrest of growth in G,. Brailovsky et al. (1973) showed that the addition of glycolipids that decreased the growth of transformed cells resulted in an elevation of CAMP. Gazdar et ul. (1972) reported decreased growth of MSV-transformed 3T3 cells and Balb-3T3 cells in response to BtcAMP treatment. Wright (1973) showed an inhibitory effect of sodium butyrate on the growth of CHO cells, a response that in other systems has been shown to result from increased CAMP levels (Prasad ct al., 197311). Yoshikawa-Fukada and Nojima (1972) noted higher CAMP levels in 3T3 cells than in their SV40transformed derivatives. Sakiyama and Robbins ( 1973) reported the inhibition of growth of normal and sarcoma virus-transformed derivatives of hamster embryo fibroblast (Nil) cells by Bt,cAMP. Walters et al. (1974) reported an arrest in growth in GI in CHO cells treated

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with caffeine. Sandor (1973) showed reversible growth inhibition by Bt,cAMP of rhabdomyosarcoma cells grown in soft agar or on a substrate. De Asiia et al. (1973) showed that Bt,cAMP prevented the stimulation by insulin of growth of BHK cells in agar culture. Responses to CAMP of cells grown in suspension (spinner) culture have also been studied. Schroder and Plagemann (1971) reported no change in growth with CAMP treatment of rat hepatoma, L, or HeLa cells in suspension culture. However, Oler et al. (1973) showed inhibition of growth of L cells in suspension culture with Bt,cAMP treatment. Inhibition of L-cell growth in spinner culture has been observed with Bt,cAMP by others (G. S. Johnson, unpublished observation). The picture that evolves from these nunieroiis studies on CAMP and growth control (reviewed earlier by Pastan et al., 1974) is one of a central regulatory function for CAMP. Measurement of CAMP levels in response to growth stimulation or inhibition by various agents shows that high CAMP in these fibroblastic, embryonic-type cells is coupled with growth arrest in both normal and transformed cells. The cell cycle points at which growth ceases vary in predominance from cell to cell, but in general two major points are under CAMP control: a point in early G, and one in G2. More significantly, perhaps, is that these changes in CAMPare probably not coincidental or concurrent with other events that control growth, since treatment of varoius types with agents that relatively selectively raise or lower CAMP result in growth control at exactly these same points. Even though this regulatory system may have other parts, particularly in more highly differentiated cells (involving perhaps cGMP), there is overwhelming evidence that growth regulation in these spontaneously growing fibroblastic cultured cells is mainly dependent on CAMP. Furthermore, the critical question of the defect that is induced in growth control following malignant transformation appears to b e in many cases reflected in defects of CAMP metabolism (Pastan et d.,1974). Herein lies a major hope in the understanding of the nature and eventual manipulation of malignant growth.

B. CELLS OF NEURONAL ORIGIN The growth of neuronal cells in culture follows the same general pattern of response a s that of embryonic or fibroblastic cells. I n general, CAMP appears to be an inhibitory regulator of cell growth. The experiments on neuroblastic cells have an added facet in that CAMP h a s been proposed to be a stimulator of a differentiated response at the same time it inhibits growth. As a result, neuroblastoina cells

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treated with BtcAMP are said to show both morphological differentiation and growth inhibition, both of which become relatively irreversible after a few days. This property of induced irreversible differentiation by cAMP seems to be unique to neuroblastoma. The most extensive studies of neuroblastoma have been those of Prasad and his collaborators who showed that mouse neuroblastoma cells rapidly slow growth after BtcAMP treatment and fail to reinitiate growth in normal medium if BbcAMP is removed after 3-4 days (Prasad and Hsie, 1971). This cessation of growth is accompanied by decreased DNA synthesis (Prasad et al., 1972). Human neuroblastoma cells were shown to respond similarly with the same irreversible cessation of growth (Prasad and Mandal, 1972, 1973). Other agents also decreased growth (sodium butyrate, 5’-AMP, ATP, ADP) but failed to show morphological differentiation (Prasad and Vernadakis, 1972). The growth arrest of BbcAMP-treated cells appeared to occur in the GI phase of the cell cycle, as judged by decreased DNA per cell content (Prasad et al., 1973a). Others have shown growth inhibitory effects of Bt2cAMP on neuroblastoma cells (Furmanski et al., 1971; Hamprecht et al., 1973; Lim and Mitsunobu, 1972), or decreased growth after treatment with agents that elevate cAMP levels such as PGEl and theophylline (Gilman and Nirenberg, 1971). It has also been reported that human neuroblasts show a relatively irreversible differentiation reaction to Bt2cAMP (MacIntyre et al., 1972). Other neuronal tumor cells also are inhibited in growth by Bt2cAMP, although not irreversibly. Human tumor astrocytes (MacIntyre et al., 1972), rat glioma (Hamprecht et al., 1973), and rat astrocytoma cells (Lim and Mitsunobu, 1972) are all inhibited b y Bt2cAMP. C. EPIDERMAL CELLS Voorhees and his collagues studied the role of cAMP in epidermal cell proliferation. The initial observations related to the ability of catecholamines to inhibit cell proliferation in intact epidermis (Powell et al., 1971). By measureing cAMP levels in skin, it was found that the epinephrine-induced decrease in epidermal mitosis was accompanied b y an increase in cAMP (Voorhees et al., 1972a; Bronstad et al., 1971). Further, BbcAMP was shown to inhibit specifically cell proliferation in epidermis, a reaction absent with sodium butyrate or 5’-AMP (Voorhees et al., 1972b). This inhibition of proliferation was also shown with cAMP (Marks and Rebien, 1972). Of specific interest in these studies, however, was the eventual im-

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plication of defective CAMP metabolism in proliferative skin diseases, notably psoriasis. By measuring CAMP levels in psoriatic le) that CAMP levels were lower in sions, Voorhees e t d.( 1 9 7 2 ~ found psoriatic skin as compared with uninvolved skin of the same patient, or the skin of normal individuals (Voorhees et al., 1972b). Since glycagon content of psoriatic skin was high, and the proliferative rate in these lesions was also high, Voorhees suggested that a basic problem with psoriatic lesions was evident in their low CAMP levels (Voorhees, and Duell, 1971). By further measurements, it was subsequently shown not only that CAMP levels were low, but that cGMP levels were higher in these lesions (Voorhees et al., 1973a), supporting the postulate of an inverse relationship between these cyclic nucleotides in regulating cell growth (Goldberg et ul., 1974). This subject has been extensively reviewed by Voorhees and his colleagues (Voorhees and Mier, 1974; Voorhees e t d., 1973b,c, 1974). The significance of this group of studies is enhanced by the possibility of treating proliferative skin disorders with agents that elevate CAMP levels.

D. MISCELLANEOUSCELLS Numerous studies have appeared which demonstrate the ability of CAMP analogs, usually Bt,cAMP, or agents which elevate CAMP levels, to inhibit the growth of cells in culture. Many of these studies utilize specialized cell lines or organ cultures. Almost all show reversible growth inhibition in response to treatments that cause increased CAMP. Cells of muscle origin that have shown this growth inhibition by CAMP are an established myogenic cell line (Wahrmann et al., 1973), primary chick myoblasts (Zalin, 1973), and human rhabdomyosarcoma cells (Sandor, 1973). Cells of lymphoid or leukemoid origin that have shown growth inhibition due to CAMP include a transformed lymphocytic cell line (RPMI-1788) (Werthamer et al., 1974), another malignant lymphoid cell line (RPMI-8866) (Millis et al., 1972), mouse leukemia cells (L-5178-Y-R) (Yang and Vas, 1971), and a plasma cell tumor in suspension culture (Naseem and Hollander, 1973a,b). Epithelial cells have also demonstrated growth inhibition due to CAMP, for example, KB cells (Teel and Hall, 1973), HeLa cells (Kaukal et ul., 1972), hamster cheek pouch tissue (Teel, 1972), and rat lens epithelium (Grimes and von Sallmann, 1973; von Sallmann and Grimes, 1974). The growth of cells derived from liver has also been shown to be

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influenced by cAMP (Van Wijk et al., 1972, 1973; Nose and Katsuta, 1974). Melanoma cells have been reported to be inhibited by cAMP (Wong and Pawelek, 1973; Kreider et al., 1973). Mastocytoma cells (Keller and Keist, 1973) and adrenal tumor cells (Masui and Garren, 1971) were also inhibited by CAMP. Uncloned chick embryo fibroblasts (CEFs) do not show the same alterations in growth patterns or growth rate with transformation seen in mouse, rat, or human fibroblastic cells. The response of these cells to cAMP treatment may thus not be the same. Hovi and Vaheri (1973) claimed a stimulatory effect on growth of cAMP and cGMP at low concentrations in primary CEFs. The role of cAMP in the initiation of the immune response and in inflammation, or its effects on immunological transformation and immune recognition, are beyond the scope of this article. The function of cAMP in these highly complex reactions and the roles of calcium and cGMP have been reviewed and summarized elsewhere (Whitfield et al., 1973; Watson et al., 1973; Hadden et al., 1972; Braun et al., 1974; Webb et al., 1973; Weissman et al., 1971, 1972; Bourne et al., 1974).

E. EFFECTS ON THE CELL CYCLE cAMP controls the growth of numerous cell types, as discussed in this article. A central question existed, however, whether this inhibitory control was a general slowing of all cell processes and all phases of the cell cycle, or whether this control was specific for specialized points during the cell cycle. This would hopefully lead to further understanding of the exact mechanism by which cAMP controls growth. Relevant studies were undertaken by numerous investigators often using different cell lines. Figure 6 represents a model of the points during the cell cycle that are under the influence of CAMP. The largest areas of agreement suggest that there is a point, sometimes referred to as a restriction point (Pardee, 1974) for growth control in early GI. Prior to this point, cAMP acts as an inhibitory controller of the cell cycle, preventing cells from passing this restriction point. This has been demonstrated in 3T3 mouse cells (Willingham et al., 1972; Bombik and Burger, 1973; Kram et al., 1973; Burger et al., 1972b; Schor and Rozengurt, 1973), in embryonic rat cells (Frank, 1972), in human diploid fibroblasts (Froehlich and Rachmeler, 1972, 1974), in BHK cells (Pardee, 1974; Zimmerman and Raska, 1972), in CHO cells (Remington and Kelvecz, 1973; Rozengurt and Pardee, 1972), in transformed lymphoid cells (Smets, 1973), in KB cells (Tee1 and Hall, 1973), and in neuroblastoma cells (Prasad et al., 1973a).

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M

FIG.6. A summary of the effects ofcAMP on phases of the cell cycle (see Section

111,E).(From Pastan et u l . , 1975.) Reproduced, with permission, from “The Role of

Cyclic Nucleotides in Growth Control” by I . Pastan, G. S. Johnson, and W. B. Anderson, Anrtuol Reaieic; of Biocheniistry, Volume 44.Copyright @ 1975 by Annual Reviews Inc. A11 rights reserved.

After this restriction point has passed, some, but not all, cells show enhancement of progression into DNA synthesis with cAMP treatment (Willingham et nl., 1972). A second point of inhibitory control appears prior to mitosis in G2. This has been shown in 3T3 cells (Willingham et al., 1972), SV40-3T3 cells (Smets, 1972), HeLa cells (Zeilig et al., 1972), epidermal cells (Marks and Rebien, 1972), CHO cells (Remington and Klevecz, 1973), and human lymphoid cells (Millis et al., 1972). Once this G, restriction point has been passed, cAMP has been reported to cause a more rapid progression out of mitosis in HeLa cells (Zeilig et d.,1974). The relatively selective restriction of cell cycle progress in G, by CAMP has been suggested to be the explanation for the GI arrest of contact-inhibited cells with rising CAMP levels due to cell contact (Willingham et ul., 1972). This concept is supported by the observation that agents that release cells from contact inhibition also result in falling CAMP levels, such as proteases, serum, or insulin (Burger et nl., 1972b; Seifert and Rudland, 1974; Otten et al., 197213).

IV. Effects of cAMP on Biochemical Functions A.

MEMBRANETRANSPORT

The ability of the plasma membrane of cultured cells to transport specific molecules is a relatively easily measured function if these molecules are available in a radioactively labeled form. As a result,

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transport processes were measured after malignant transformation i l l zjitro by many investigators. These experiments led to the use of altered transport as a measure of transformation. In general, malignant transformation results in more rapid transport of low-n; olecularweight nutrients, particularly glucose. Since CAMP appeared to alter other properties of transformation, its effects on transport of nutrients, especially amino acids, nucleotides, sugars, and phosphate, were measured after the use of treatments or agents that raise CAMP levels. Grimes and Schroeder (1973) reported that treatment of either normal or Py-transformed 3T3 cells with BtcAMP lowered the ability of cells to transport 2-deoxyglucose. 3T3 cells in either sparse or confluent culture transport 2-deoxyglucose at a lower rate than that for transformed derivatives (Schultz and Culp, 1973). The lower CAMP levels in transformed cells might then be related to higher rates of glucose transport. However, Gazdir et al. (1972) initially reported a stimulation of glucose uptake with prolonged BtcAMP treatment of Balb-3T3 cells. The increased glucose transport following transformation has been shown in one system to require new protein synthesis (Bader, 1972). Other alterations in morphology or CAMP levels in this system are not affected by inhibiting protein synthesis. This implies that glucose transport may be a function of transformation that occurs much later and is not immediately under the control of CAMP. Indeed, experiments with the 3T3 cAMPtCSmutant (Willingham et d.,1973) have shown that 2-deoxyglucose uptake does not change in the first 15 minutes after temperature shift and falling CAMP levels (M. C. Willingham, unpublished observations). The control of transport of other nutrients appears more clear-cut. Uridine transport has been shown to be decreased by Bt,cAMP or PGE, (Hauschka et al., 1972; Kram et al., 1973). An increase in uridine transport after serum or insulin treatment (De Asua et UZ., 1974; Hershko et al., 1971) was blocked by raising CAMP (Rozengurt and De Asua, 1973). An increase in transport of leucine after serum addition was prevented by BtcAMP (Paul, 1973). Leucine transport in 3T3 cells was decreased b y PGE, treatment (Kram et al., 1973).Rozengurt and Pardee (1972) reported a decrease in aminoisobutyrate and glutamine transport following BtcAMP treatment. Kram and Tomkins (1973) showed that some of the inhibitory effects of CAMP on transport were reversed by the inclusion of cGMP after the addition of CAMP, a phenomenon also observed with Colcemid or vinblastine treatment. It is not clear whether these were

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drug effects on membranes directly, or if these reversals of transport inhibition actually operated through intracellular functions such as cGMP levels or effects on niicrotubules. Phosphate transport has also been studied with CAMP. Blat et nl. (1973) reported a decrease in phosphate transport after Bt,cAMP treatment in BHK cells. De Asua et al. (1974) demonstrated that an immediate increase in phosphate transport in 3T3 cells after new serum addition was accompanied by falling CAMP levels. Mouse embryo fibroblasts showed the same relationship (Rozengert and De Asua, 1973), but it was pointed out that the increase in phosphate transport was not as readily reversible by PGE, or theophylline, suggesting that it might not be under direct control of CAMP. In the context of the postulated mechanism of a pleiotypic response presented by Hershko et al. (1971),CAMP has some of the characteristics of a pleiotypic mediator. It appears eventually to inhibit the transport of amino acids and nucleotide precursors. What is not clear is how intimate the association of cAMP is with these responses, or its role in the transport of other nutrients such as glucose or phosphate. It is only possible to suggest that, after the changes in cell function occur with prolonged CAMPtreatment, there are accompanying alterations in membrane transport functions, some of which are possibly secondary to overall alterations caused primarily by CAMP.

B. ENZYMEINDUCTION BY cAMP

The activities of specific enzymes in numerous systems are altered by cAMP fluctuations. Most of these studies have dealt with the in-

duction of new enzyme production, either of enzymes involved in hormonal reactions mediated through CAMP, or changes in the enzymes of CAMP metabolism itself. D’Armiento et nl. (1972) showed that the enzyme that degrades cyclic nucleotides, CAMP PDE, is induced b y raising cAMP levels. This implied that CAMP could induce the production of its own degradative enzyme and thus regulate excessively high CAMP levels. This induction occurred with Bt,cAMP treatment of 3T3 cells and PGE, treatment of L cells. The induction was prevented by treatment with cycloheximide or actinomycin D, indicating transcriptional control of new message synthesis b y CAMP. Maganiello and Vaughan (1972) showed a similar induction of PDE in L cells with PGE, treatment. Schwartz et al. (1973) showed induction of PDE after Bt,cAMP treatment of glial tumor cells. Anderson et (11. ( 1 9 7 3 ~ showed ) increases in PDE activity as a re-

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sult of cell density, along with increasing adenylate cyclase activity. What was most interesting, however, was that contact-inhibited cells stopped increasing PDE activity at the time of cell contact, whereas cyclase activity continued to rise. As a result, CAMPlevels rose, producing at confluency the inhibitory control of CAMPseen in contact inhibition. However, chick embryo cells grown under conditions in which contact inhibition does not occur failed to prevent the rise in PDE, and CAMP levels remained low, allowing continued growth beyond confluency. Thus contact inhibition might be regulated initially by alterations in PDE activity with cell contact. Increased CAMPlevels or BbcAMP has been shown to induce new enzyme synthesis in enzymes of neural transmitter pathways in neuronally derived cells. Tyrosine hydroxylase activity has been shown to increase in neuroblastoma cells after BtcAMP treatment (Waymire et al., 1972; Richelson, 1973; Prasad et al., 1972). BtcAMP treatment of neuroblastoma cells also increased the activity of acetylcholinesterase (Furmanski et al., 1971; Prasad and Vernadakis, 1972) and choline acetyltransferase (Prasad and Mandal, 1973), but not catachol-0-methyltransferase (Prasad and Mandal, 1972). In fetal rat brain cells, BtcAMP increased acetylcholinesterase (Shapiro, 1973) and glutamate decarboxylase (Schrier and Shapiro, 1973). Wong and Pawelek (1973) showed increased tryosinase activity in melanoma cells after BtcAMP or MSH treatment. Cells of liver origin have shown inducible enzymes with BtcAMP treatment. In Reuber hepatoma cells, phosphoenolpyruvate carboxykinase (Bamett and Wicks, 1971) and tyrosine aminotransferase (Barnett and Wicks, 1971; Butcher et d., 1971) are increased by BbcAMP treatment. In other hepatoma cell lines, phenylalanine hydroxylase (Haggerty et al., 1973) and tyrosine aminotransferase (Stellwagen, 1972; Grossman et al., 1971) increased after BtcAMP treatment. In other cultured liver cells, glucose-Gphosphatase (Verne et al., 1973) and alkaline phosphatase (Nose and Katsuta, 1974) increased after BtcAMP treatment. Alkaline phosphatase also increased after BtcAMP treatment in a hybrid cell line (Koyama et al., 1972). In addition to cells of neuronal or liver origin, CHO cells increased their activity of serine dehydratase with BtcAMP treatment, mainly in late S phase in synchronized cells (Kapp et al., 1973). C. PRODUCTION OF CELL PRODUCTS BIOCHEMICAL FUNCTIONS Studies of the ability of cells to release or synthesize specific cell products, and its modulation by CAMP, have appeared sporadically in the literature. In cases in which the product is related to hormonAND OTHER

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ally mediated specific synthesis, CAMP’S mediation of these pathways is usually evident in increased production with CAMP or Bt,cAMP treatment in the absence of the hormone. Differentiated functions of cultured cells such as mucopolysaccharide or hyaluronic acid synthesis of mesenchymal cells are stimulated by CAMP. Among the hormonally mediated roles of CAMP are steroid production increases in adrenal or adrenal-derived cells after CAMP treatment (Sayers et al., 1972; Kowal, 1970, 1973; O’Hare and Neville, 1973b), the release of glucose from hepatocytes after BhcAMP treatment (Verne et al., 1973), the release of progestins in bovine luteal cells (Gospodarowicz and Gospodarowicz, 1972), and the stimulation of granule pseudopodia (and thus enzymic release) in rat parotid gland (Schramm et al., 1972). However, CAMP appears to be inhibitory to the release of lysosoma1 granules in leukocytes (Zurier et al., 1973; Goldstein et al., 1973), and to inhibit the release of cytotoxin in lymphocytes (Lies and Peter, 1973). Mucosubstance and hyaluronic acid synthesis have been shown to be stimulated by Bt,cAMP treatment. This has been reported for mucosubstances in 3T3 and SV40-3T3 cells (Goggins et d., 1972), hyaluronic acid synthesis in human synovial cells (Castor, 1974), mucosubstance secretion in Rous sarcoma cells (Coe et al., 1970), and collagen synthesis in CHO cells (Hsie et al., 1971). The glycogen content of HeLa cells has been reported to decrease after treatment with BhcAMP (Hilz and Tarnowski, 1970; Kaukel et al., 1972). Isolated mature liver cells showed decreased lipid synthesis with BhcAMP treatment (Capuzzi et al., 1974). The phosphorylation of plasma membrane components in CHO cells has been reported to alter after BhcAMP treatment (Rieber and Bacalao, 1973). Other overall cell functions such as DNA, RNA, and protein synthesis may be altered by BhcAMP treatment (Lim and Mitsunobu, 1972). Since CAMP alters the length of or entry into various phases of the cell cycle (Section II1,E) it can change the overall content of biochemical components whose levels are dependent on phases of the cell cycle (DNA, RNA, or protein).

V. Properties Mediated through the Cell Surface A.

MOTILITY

AND

MIGRATION

Cultured cells often show extensive motility on glass or plastic substrates. The exact mechanism by which this process occurs is not entirely clear, but the involvement of microfilamentous and microtu-

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bular cellular elements is quite striking, as well as the ability of cell structures to show active contraction and cell body translocation (Abercrombie et aZ., 1973). The involvement of cAMP in this process has been noted, but the actual mechanisms by which regulation of motility occurs are not completely clear. A postulate relating the contractile elements of cells and cAMP has appeared (Willingham and Pastan, 1975a) (see Fig. 5). In this proposal cAMP in fibroblastic cultured cells is viewed as an inhibitory regulator of microfilamentous function, and thus cell process contraction, while being a stimulatory regulator of microtubular assembly, along with the ability of cAMP to increase adhesiveness. The previous literature relates two different types of roles for CAMP. One is the ability of cAMP to be a positive chemotactic attractant for leukocytes. The other involves the general effects of cAMP on the motility mechanisms of cells when it is raised intracellularly throughout the entire cell. Leukocytes and macrophages are highly motile cells in uiuo, their rate of motion being far greater than that shown b y fibroblastic cells in culture. Yet it appears that cAMP may play a similar role in the overall regulation of the ability of cells to move. In experiments designed to evaluate the effect of raising cAMP on the chemotactic or migratory response of these highly motile cells, cAMP appeared to b e an inhibitor of migration (Rivkin and Becker, 1972; Estensen et al., 1973; Pick, 1972). Bore1 (1973)disagreed with these results in observing the migration of rabbit neutrophils retrieved after injecting an irritant into the peritoneal cavity. Koopman et aZ. (1973) suggested that the inhibitory ability of migration inhibitory factors (MIF) on macrophage migration was not due to cAMP as suggested by Pick (1972), since Bt,cAMP or PGEl interfered with MIF activity and these agents did not seem to affect migration directly, Fibroblastic cells, although they move more slowly than leukocytes, can still show active motility by time-lapse cinematography. Johnson et al. (1972) showed that BtcAMP or PGEl inhibited the motility of L929 cells in culture. Smets (1972) reported that SV40-3T3 cells were inhibited from migration into a monolayer scratch wound. Willingham et aZ. (1973) reported the control of cell process retraction in a 3T3 cAMPtcsmutant. Falling cAMP levels resulted in process retraction, whereas high cAMP prevented this response. Using this example and other morphological evidence, Willingham and Pastan (1975a) have postulated a cAMP control mechanism for fibroblastic cell motility. The other area of interest concerning cAMP and migration comes

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from the observations that CAMP, BtcAMP, and PGE, are chemotactic attractants for neutrophilic leukocytes (Leahy et al., 1970; Kaley and Weiner, 1971; Gamow et al., 1971; Grimes and Barnes, 1973a,b; Gamow and Barnes, 1974). Moreover, cGMP appeared to be a partial inhibitor or repelling agent for chemotaxis (Gamow and Barnes, 1974). Thus it acted in a manner opposite CAMP,a result similar to the stimulation of overall migration to other chemotactic factors by cGMP, since CAMP was inhibiting for this action (Estensen et al., 1973).The overall meaning of these results is not entirely clear, but one could imagine that, in a gradient of CAMP, the changes in substrate adhesiveness caused by local CAMP elevations might result in overall cell migration toward that source. However, elevating total cell CAMP high enough in a nondirected fashion would inhibit the migratory apparatus of the cell and thus slow down overall migratory activity. B. AGGLUTINATION BY PLANT LECTINS A major difference determined between normal and transformed cells is the relative lack of agglutination with plant lectins in normal cells, but the marked agglutination seen after transformation. Prolonged treatment of transformed cells with Bt,cAMP or PDE inhibitors decreased their agglutinability with the plant lectins Con A or wheat germ agglutinin (Hsie et ul., 1971; Sheppard, 1971; Korinek et al., 1973; Kurth and Bauer, 1973; Prasad and Sheppard, 1972). In one study (Tihon and Green, 1973), the same cell type (CHO-K1) examined by Hsie et al. (1971) showed increased agglutination with Con A with BtcAMP treatment, but the simultaneous release of RNA viral particles produced by Bt,cAMP treatment may have altered the cell surface in a manner not strictly due to increased CAMP. There were two major difficulties, however, with these studies. All involved prolonged treatment with an external drug, and there was no easy way to quantitate the changes observed. Smets (1973) pointed out that, in cells whose agglutination was apparently unaffected by Bt,cAMP treatment (EB-virus-transformed lymphoid cells), changes in agglutinability occurred corresponding to phases of the cell cycle, the lowest being in G,. Since prolonged BtcAMP treatment synchronized SV40-3T3 cells in G,, he then proposed that the decreased agglutination they showed might be due to cell cycle synchrony. Recently, however, the role of CAMP in agglutination was clarified by a study in which a quaiititation method for Con-A agglutination was devised (Willingham and Pastan, 1974). In this study a mutant of 3T3 cells with CAMP metabolism sensitive to temperature change

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was used to eliminate external agent influences. It was shown that, with rapidly falling cAMP levels, these cells rapidly became agglutinable. Preventing this fall in cAMP by a short (15minute) preincubation with various agents that raised cAMP levels prevented the increases in agglutinability. Similar short preincubations in a transformed cell (L929) produced similar, rapidly reversible, decreases in agglutinability. This demonstrated that agglutinability changes were not due to cell cycle synchrony, because of the short times involved, and that agglutination could be seen even in normal cells when cAMP levels were lowered. The exact mechanism by which cAMP regulates agglutination was not clear, but Willingham and Pastan (1975b)found that raising cAMP levels can directly reduce the presence of surface microvilli in L929 cells, and that 3T3 cAMPtCSmutant cells suddently emit multiple microvilli in response to the falling cAMP levels after temperature change. They further showed the direct involvment of microvilli in mediating the formation of cell clumps during lectin agglutination. I t seems likely therefore that agglutinability b y plant lectins is mediated through cAMP regulation of cell surface microvilli, and that the presence or absence of these microvilli is one of the main reasons for the agglutination difference between transformed and normal cells in culture.

c.

ADHESIVENESS AND OTHER CELL SURFACE PROPERTIES The ability of cAMP to alter cultured cell morphology and motility may be in part related to its effects on cell-to-substrate adhesiveness (Willingham and Pastan, 1975a). When cultured cells are treated with agents that elevate CAMP, they are more difficult to remove from their substrate by protease or ethylene glycol bis (p-1 aminoethyl ether)-N,N’-1 tetraacetic acid (EGTA) treatment (Johnson and Pastan, 1971, 1972c; Prasad and Hsie, 1971; Grinnell et al., 1973; Gazdar et al., 1972). This was reported for both normal and transformed cell lines (Gazdar et al., 1972). This “detachability” of cells differs from measurements of the initial adhesion rate of either lowadherence or protease-treated cells. Initial adhesion has been reported to be relatively unaffected by BtcAMP treatment in BHK cells (Grinnell et al., 1973), whereas other studies have claimed a decrease in initial adhesion rate with various cyclic nucleotides (Weiss, 1973). Whatever the effects on initial adhesion, it seems clear that high cAMP levels are associated with firm adherence to the substrate in cells that have become attached. Willingham et al. (1973) reported

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that when CAMP levels fall in a mutant 3T3 cell with CAMP metabolism sensitive to temperature change, there is an accompanying fall in adhesiveness measurable by mechanical removal with medium spraying. Preincubation with agents that prevent this fall in cAMP prevented the loss of adhesiveness, The loss of substrate adhesiveness, however, had little or no effect on cell-to-cell adherence, which apparently operates through some other mechanism. Other changes due to cAMP have been reported for other surface properties. In examining cell surface antigens in virally transformed cells, Gazdar et (11. (1972) reported that BtcAMP did not seem to alter surface expression of antigens associated with transformation. However, Kurth and Bauer (1973) extensively studied the changes in surface antigen expression in a transformed cell line (DJ. Tumorspecific surf’ace antigens seemed to be increased after Bt,cAMP treatment, as were embryonic antigens. However, normal xenogenic cell surface antigens were present in smaller amounts. Therefore, the reversal of morphological appearance due to BtcAMP does not seem to b e accompanied by a reversal of the expression of transformation antigens , Another area of study has been the alterations in cell surface components that change during transformation. Baig and Roberts (1973) and Roberts et ul. (1973) reported a quantitative change in the membrane distribution of surface glycopeptides. Brailovksy et ul. (1973) reported that treatment of transformed cells with certain glycolipids could change their growth pattern back toward normal. Such treatment in one case was associated with rising CAMP levels.

VI. Malignancy and Differentiation A.

MALIGNANTTRANSFORMATION I N CULTURED CELLS AND TUMORIGENICITY

Many properties distinguish malignant transformation in culture. Among these are faster logarithmic growth rate, loss of densitydependent inhibition of growth, ability to grow in soft agar, ability to grow in low concentrations of serum, rapid motility, low adhesiveness, faster uptake of many nutrients, round shape, and high agglutinability with plant lectins. Not all of these properties are altered in the same fashion in all cell types. Since transforming viruses have only a small amount of genetic information, it might be expected that they would produce these varied results by affecting some common

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intracellular regulatory molecule (e.g., CAMP). Almost all of these general properties have been shown to be modulated in some way by CAMP. We have dealt with these individual effects in previous sections. In general, cAMP reverses the effect of transformation on many properties, returning them toward normal. These changes have also been summarized elsewhere (Pastan et al., 1974; Pastan, 1972). In this section we deal with the role of cAMP in the transformation process, and also its usefulness as an in wiwo carcinostatic agent. It has been shown that, in many in vitro transformation systems, cAMP metabolism is altered. T h e levels of cAMP are usually lower in transformed derivatives than in their parent cell lines (Otten et al., 1971; Sheppard, 1972). In the transformation of embryo cells with RSV, Otten et al. (19724 showed that morphological transformation was preceded by a fall in intracellular CAMP. This transformation could be prevented by maintaining cAMP high with BtcAMP. Anderson et al. (1973a,b) later showed that the activity of adenylate cyclase fell preceding this fall in cAMP with temperature-sensitive mutants of both Bryan (Anderson et d.,1973a) and Schmidt-Ruppin (Anderson et al., 1973b) strains of RSV (also see Russell and Anderson, 1973). Further, Anderson et d.(1973~) showed the relationship of adenylate cyclase, CAMP, and PDE to density-dependent inhibition of growth in NRK cells, and that this mechanism for cAMP control is not present in C E F grown under conditions in which they are not density-inhibited for growth. Thus a major property that fails in transformation, density-dependent growth control, seems to b e mediated through CAMP. In addition, Carchman et al. (1974) showed that a temperature-sensitive mutant of MSV produced a fall in cAMP accompanying transformation, similar to the RSV system, although this temperature-sensitive MSV requires 2 4 4 8 hours to produce transformation. From these studies it seems clear that viral transformation of fibroblastic cells in culture is intimately associated with defective cAMP metabolism. Other studies have dealt with the effects on cAMP of transforming viruses during initial infection. It is not clear that these changes actually relate to the transforming ability of these viruses, since the infections either lead to cell death rather than transformation, or to a turnon of cellular DNA synthesis in the absence of transformation in most of the cell population. Rein et al. (1973) showed that cAMP levels fall after infection of Balb-3T:3 cells with SV40 virus, preceding an increase in cellular DNA synthesis. Raska (1973) reported that infection of BHK cells with adenovirus reduces cAMP levels and lowers adenylate cyclase activity. Zimmerman and Raska (1972) showed an inhibition of induced cellular DNA synthesis after adenovirus infection by BtcAMP.

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Another role of cAMP in the transformation process deals with the effect of CAMP on the actual transforming efficiency of oncogenic viruses. BHK cells infected with Py showed increased transformation frequency with Bt,cAMP added 24 hours after infection (Smith et al., 1973). In the same study, the transformation frequency of CHO cells infected with SV40 virus increased with Bt,cAMP treatment during the S phase of the cell cycle. The production or replication of viruses has been studied with CAMP treatment. Tihon and Green (1973) showed that Bt,cAMP treatment of CHO cells resulted in increased production of RNA tumor viruslike particles. Biron and Raska (1973) reported decreased adenovirus replication after Bt,cAMP treatment in fibroblastic cells. Bt,cAMP was shown to have little effect on the replication of murine leukeniia virus (MuLV) in Kirsten sarcoma virus-transformed 3T3 (Gazdar et al., 1972), and it fails to induce MuLV in AKR (mouse embryo) cells (Teich et al., 1973). Since CAMP metabolism appears defective in many transformed cells and many transformed properties could b e reverted toward normal with CAMP treatment, many studies have investigated the usefulness of cAMP as a therapeutic tool for malignancies in wiuo. Unfortunately, most of the cell types studied in intact animals are not ones in which cAMP metabolism has been well characterized in culture. The measurement of cAMP in tumor tissue has not demonstrated remarkably low levels of CAMP. Chandra and Gericke (1972) reported increased cAMP levels in endocrine tumors, and E. W. Thomas et al. (1973) reported high cAMP levels in hepatomas compared to normal liver. In spite of these results, numerous studies have appeared which attempt to decrease tumor growth by either directly treating transplanted tumors in animals with CAMP, or pretreating tumor cells in culture with cAMP prior to transplantation. Some of these studies have shown significant effects. Decreased tumor growth after cAMP treatment was reported for lymphosarcoma (Chandra and Gericke, 1972; Chandra e t al., 1972; Gericke and Chandra, 1969), CELO virus-transformed hamster cells (Reddi and Constantinides, 1972), IU3 cells in hamster cheek pouch (Smith and Handler, 1973), MPC-11 plasmacytoma in mice (Naseem and Hollander, 1973a,b), and Ehrlich ascites tumor cells (Seller and Benson, 1973a,b). Cho-Chung and Guillino ( 1974a,b) showed the inhibition of growth for both Walker carcinoma and rat mammary tumors after Bt,cAMP treatment. IN CULTURE B. DIFFERENTIATION Elevating cAMP levels in cultured cells often results in increased expression of characteristics that have been associated with cellular

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differentiation. Whether CAMP plays this role in uiuo, or is a common mediator of differentiated properties, is not known. Cells of neuroectodermal origin appear to be relatively sensitive to CAMP as an inducer of cellular differentiation. True differentiation is probably best seen in systems in which the differentiated state remains after the inducing agent has been removed. The ability of CAMP to cause this process has been shown for both neuroblastoma and melanoma cells in culture. CAMP or agents that increase intracellular CAMP can induce irreversible morphological differentiation in mouse and human neuroblastoma cells in culture (Prasad and Hsie, 1971; Prasad and Vernadakis, 1972; Furmanski et al., 1971; Prasad, 1972; Prasad et ul., 1972, 1973a,b; Prasad and Sheppard, 1972; MacIntyre et al., 1972; Prasad and Mandal, 1972, 1973). Changes in the direction of differentiation have been reported in sensory ganglia (Roisen e t al., 1972a,b). Melanoma cells undergo changes perhaps analogous to differentiation through increased pigment production (Johnson and Pastan, 1972b; Wong and Pawelek, 1973; Kreider et al., 1973). Other cell types show changes toward differentiation after CAMP treatment. Channing and Seymour (1970)reported changes similar to LH or FSH effects in granulosa cells in culture after Bt,cAMP treatment. The production of antigenic material specific for cervicovaginal epithelium was increased after CAMP treatment of cervicovaginal anlage from neotal mice (Fjellested and Kvinnsland, 1971). A measurement of differentiation in cultured muscle cells, the fusion of cells into myotubes, has been examined after BtcAMP treatment. In a myogenic cell line, Wahrmann et a1. (1973) reported decreased myotube formation after BbcAMP treatment. Zalin (1972, 1973) reported delayed fusion of primary chick myoblasts after Bt,cAMP treatment. It appears therefore that in normal muscle cells high CAMP levels may interfere with myotube-cell fusion. However, the response of malignant muscle cells appears to be toward differentiation after CAMP treatment, as reported by Aw et al. (1974) in a transformed muscle cell line (16A).

VII. Concluding Remarks

The studies reviewed in the preceding sections have made it clear that CAMP regulates or affects a great number of the properties of cells that are measurable in culture. We have concluded that the multiple effects of CAMP probably indicate that nature has used this molecule for regulatory functions rather frequently. The ubiquitous

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occurrence of CAMP from bacteria to mammalian cells, and the common thread of cAMP as a regulator of specific functions in different cell types, points out the uniqueness of its role. The wide variety of reactions of cells from different origins has led to the concept that cAMP is capable of turning on those mechanisms for which the cell has been genetically programmed. How does one reconcile this with the ability of cAMP to slow the growth of cultured cells? Perhaps these cells required cAMP to control their proliferation on the way to differentiation but, after their genetic machinery has been committed for a specific purpose, cAMP is used as a regulator of the expression of specific differentiated functions. When these cells undergo malignant change, however, there sensitivty to cAMP control of growth reappears, and only those cells that can overcome this control go on to produce tumors. This last idea raises an interesting possibility. If, as much of the evidence presented here indicates, the defect in malignancy involves the failure of the cAMP growth control mechanism, then we have a “handle” with which to attack the underlying mechanisms of growth control. Beyond the interest in the control of malignancy, the understanding of the regulation of cellular events and major cellular properties will undoubtedly yield a host of important discoveries, and allow control of what are usually uncontrolled disease processes. ACKNOWLEDGMENTS The author expresses his gratitude to Drs. Ira Pastan and A. Julian Garvin for their help in reviewing this article, and to Mmes. Martha Harshman, Frances Herder, and Michele Shevitz for invaluable technical assistance in its preparation.

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