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Molecular biology of cell activation
Experimental Cell Research 183 (1989) 24-35
SPECIAL ARTICLE Molecular
Biology
LESZEK KACZMAREK**’ Departments
of *Neurophysiology Experimental Biology,
of Cell Activation and BOZENA KAMINSKA?
and TCellular Biochemistry, Nencki Institute Pasteura 3, 02-093 Warsaw, Poland
of
This article summarizes common features of activation of diierent types of cells along diierent physiological lines such as proliferation, differentiation, and execution of function of terminally differentiated cells. The common basis of many of these phenomena includes (i) first messengers (growth factors, cytokines, neurotransmitters, etc.) acting on membrane receptors, (ii) second messengers (CAMP, IPr, DAG, Ca2’) spreading an activating signal inside the cell, and (iii) elevated expression of some genes (c-fos, c-myc, omithine decarboxylase). The role of the genetic correlate in cell activation is emphasized, and it is concluded that the aforementioned genes (their protein products) should be called third messengers, whose function is mediation of long-term phenotypic changes. @ 1989 Acadetmc Press, Inc.
I. ACTIVATION
VERSUS MAINTENANCE
OF A PHENOTYPE
The term “cell activation” can be used to describe a plethora of different biological phenomena, some of which are seemingly unrelated. In this paper we suggest that the same term can designate an early phase of (predisposal to?) biological processes which have in common a long-term phenotypic change, e.g., stimulation of quiescent cells to enter the cell cycle, induction of differentiation, and long-lasting functional activity of terminally differentiated cells such as macrophages or neurones. At the beginning of our discussion we must distinguish in all of the phenomena the activation of a new phenotype from the maintenance of this phenotype. We propose that cell activation is a ubiquitous phenomenon preceding and leading to establishment of a new phenotype. In order to enter the cell cycle quiescent (Go) cells require treatment with growth factors [l]. For cell cycle stimulation normal, nontransformed cells require more than one growth factor. The best characterized cells in this regard, mouse Balb/c 3T3 fibroblasts, may serve as a model of mechanisms controlling cell proliferation [2]. There are two groups of growth factors acting in concert on those cells. Competence factors [e.g., PDGF (platelet-derived growth factor)] initiate some biochemical reactions in quiescent Balb/c 3T3 fibroblasts and in this way they prime the cells to be responsive to progression factors [EGF (epidermal growth factor) and IGF-I (insulin like growth factor I) are the most prominent examples]. Cells treated with factors belonging to both groups enter DNA synthe-
’ To whom reprint requests should be addressed. Copyright @$J1969 by Academic Press, Inc. All rights of reproductmn m any form reserved 0014-4827189 $03 00
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sis. Neither competence nor progression factors alone (at certain concentrations) suffice to stimulate the G,,-S transition of Balb/c 3T3 cells. Interestingly, the competence phase of the G& transition is present for a long time after withdrawal of the competence factors-the cells “remember” the factors they were treated with [3, 41. This means that factors responsible for the activation of the G&S transition are not required for maintenance of the active state. In cellular differentiation it is possible to clearly distinguish the induction of the terminal phenotype from its maintenance, and as in the cell cycle, differentiation inducers are very often not required to be permanently present during the transition to a new phenotype, For example, a 5-min treatment of HL60 human promyelocytic leukemia cells with phorbol esters (TPA) provokes differentiation of the cells into macrophages [5]. Similarly, differentiation of chicken myoblasts into myotubes resulting from the treatment of insulin requires only 1 min of contact between the hormone and the cells [6]. A similar phenomenon can also be observed in terminally differentiated cells. For example, neurons can be primed to increased activity by electrical stimulation, known as kindling. The enhanced neuronal excitability observed in this epilepsy-like phenomenon can still be detected well after the stimulation has been discontinued [7, 81. Also inflammation-mimicking activation of macrophages with lipopolysaccharide (LPS) drives the cells to produce cytokines even after the LPS has been removed [9]. Interestingly, all of the aforementioned phenomena of cell activation follow a similar general pathway. First, the external stimuli (first messengers) activate their specific receptors. Next, this signal is enhanced and spread inside the cells with the aid of second messengers. Finally, the specific effector systems are set up. The first messengers mentioned here (neurotransmitters, growth factors, etc.) are hydrophilic and do not easily penetrate the cell membrane. They act via their specific surface receptors and formation of second messengers. The first messengers can produce long-lasting effects often going beyond the limits of the life span of the first messenger itself. Nevertheless, the formation of the second messengers cannot explain the long duration of the signal. All of the known second messenger systems result in the activation of specific protein kinases (CAMP-dependent kinase A, Ca’+-driven kinases dependent on calmodulin, diacylglycerol-dependent kinase C, etc.) [IO-131. Therefore, their biologial effects last within the limits of the persistence of protein phosphorylation, which very often is short-lived due to a fine regulation by phosphatases [14]. However, the effects of external activation can last even for days and weeks after the stimulus application. It is widely accepted that this phenomenon can be explained by an indirect effect of first messengers on gene expression leading to the elevated production of specific mRNAs and proteins. These proteins performing their physiological role (enzymatic, structural, etc.) can in turn modify the phenotype of the cell for quite a long time. We propose that the genes encoding proteins constituting the new phenotype be designated “effector genes.” In this paper we reivew data suggesting that it is possible to distinguish also
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another class of genes, whose expression is dependent on external signals-“activation-related’genes.” While the effector genes are usually specific for the given kind of cell stimulation, the activation-related genes are at least partially expressed ubiquitously in different types of cells stimulated by a variety of biological phenomena. Moreover, their function seems to be related more to the activation than to the maintenance of a phenotype. We have selected three genes, which in our opinion best exemplify our thesis about the existence of this class of genes. II. c-myc c-myc is a cellular counterpart of the transforming gene (v-myc) of MC29, OKlO, CMII, MH2, myc-FeLV leukemic viruses [15-171. It has been detected in the genomes of all vertebrates tested so far [18]. The v-myc (viral) and c-myc (cellular) proteins are found in cell nuclei [15, 193,which leads to the suggestion that they are’ somehow involved in nuclear functions, e.g., control of gene expression. In fact, it has been shown that both v-myc and c-myc can act as transcription activators [20, 211. The c-myc protein has been found to be DNAbinding and localized to the chromatin and detected even more abundantly in the nuclear matrix 1221.On the other hand, no sequence specificity of binding of cmyc protein to DNA has been established. There is also experimental evidence for the thesis that the protein may be involved in another step in the regulation of gene expression, namely post-transcriptional processing of RNA [23]. c-myc and Cell Proliferation The most obvious support for the theory of c-myc involvement in the control of cell proliferation comes from the fact that v-myc is an oncogene. Moreover, cmyc driven by tissue-specific promoters and enhancers induces malignancies in target organs in transgenic mice [24, 253. This means that the myc gene can provide cells with a phenotype manifested by abnormal proliferation. The involvement of c-myc in normal cellular proliferation is supported by data on the pattern of its expression. The accumulation of c-myc mRNA has been observed in all kinds of Go cells stimulated to proliferate (for review see [26]). The pattern of c-myc mRNA levels always follows similar kinetics. There is an early increase, beginning at one to a few hours after application of the stimulus, the maximal levels of c-myc mRNA are reached in mid to late G& transition, and then they decline. In the second and following cell cycles the mRNA levels of cmyc are signiticantly lower than those during the G& transition and are invariant throughout the phases of the cell cycle of proliferating cells 1271. In numerous cases the factors stimulating c-myc expression have been identitied. In general, they are competence or competence-like factors, i.e., factors responsible for the early phase of the G,,-S transition. All of them act via surface receptors and second messengers. There is much information suggesting that the increase in c-myc mRNA (and protein) levels following cell cycle stimulation is not purely coincidental. Introduction of the c-myc coding sequences under the control of inducible or constitu-
Molecular
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tive promoters into mouse fibroblasts decreases the dependence of those cells on competence growth factors [28]. Similarly, microinjection of recombinant c-myc protein renders tibroblasts competent [29]. Introduction of highly expressable vmyc carrying constructs into hematopoietic/lymphoid cell lines also abrogates their dependence on growth factors [30]. Moreover, depletion of c-myc mRNA (and protein) resulting from c-myc antisense oligonucleotide treatment inhibits transition from Go to S of human T lymphocytes [31]. c-myc and Cellular Differentiation
A brief examination of c-myc expresson in differentiating cells further suggests a close link between c-myc and cell proliferation. During hemopoietic differentiation of leukemic cell lines [HL60, Friend murine erythroleukemia (F-MEL)], as well as during differentiation of teratocarcinomas and myoblasts in vitro, the decrease of c-myc expression was observed concomitantly with the declining proliferation rate of the cells [16, 32, 331. Moreover, introduction of c-myc or vmyc under constitutive promoters into F-MEL or myoblasts prevented or delayed differentiation of these cells following treatment with appropriate stimuli [34-361.
On the other hand, a closer look at the kinetics of c-myc expression in the aforementioned cells raises questions about the simple notion just described. There is indeed a dramatic decrease of c-myc mRNA levels in cells stimulated to differentiate; however, in F-MEL cells the decrease is followed by a transient rise in c-myc expression preceding terminal differentiation [37-40]. A similar phenomenon can also be observed during myogenesis [41] and differentiation of murine embryonal carcinoma cells [42]. Unfortunately, the data which present sufficiently long and detailed kinetics of c-myc mRNA levels in other cell lines are lacking. The early decrease in c-myc mRNA levels following induction of differentiation can easily be explained by the concomitant cessation of cell proliferation, as we have mentioned that c-myc is expressed, albeit at a low level, in growing cells. We interpret the following rise in c-myc expression as indicative of its involvement in the activation of differentiation. Incidentally, Caffrey et al. [36], who extensively examined differentiation of B&H1 myoblasts, observed a delay rather than an inhibition of the process following introduction of exogenous cmyc into the cells. Finally, it is noteworthy that the often-studied HL60 cells may be a particularly difficult model with which to investigate c-myc involvement in cellular differentiation. The high level of amplification of the c-myc gene in these cells [ 171probably leads to a change in the normal regulation of gene expression; hence, it is difficult to extend data obtained with this cell line to cells with normal c-myc regulation. Moreover, there are reports showing an increase in c-myc expression in cells stimulated to differentiate concomitant with the cessation of proliferation. The best-known example is rat pheochromocytoma PC12 cells treated with nerve growth factor (NGF) [43, 441. Correlation of augmented c-myc expression and differentiation has been also suggested for Purkinje cells in mouse cerebellum r451.
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In developing human embryos only a partial correlation between proliferation and c-myc expression has been noted [46-48], indicating again that not only proliferating cells are a target for c-myc biological activities. Similarly in early embryonic development of Xenopus oocytes it was difficult to correlate c-myc mRNA accumulation exclusively with cell proliferation [49]. c-myc in Terminally
Differentiated
Cells
In mouse peritoneal macrophages an increase of c-myc expression has been found following treatment with LPS, known to be an inducer of specialized inflammation-related functions of macrophages [9]. III. C-j-OS c-fos is a normal cellular counterpart of FBJ and FBR osteosarcoma transforming gene (v-fos) [26, 501. c-fos, like c-myc, is a nuclear protein [19, 501 and it has been localized to the chromatin [51, 521, where it can bind specific regions of DNA 1531,which strongly suggests its role as a gene expression regulator. In fact, both v-fos and c-fos have been shown to be able to regulate transcription [54-561. c-fos and Cell Proliferation
Again, as with c-myc, before reviewing data describing the c-fos role in the cell cycle, we must stress that it is a homolog of an oncogene. Activation of c-fos expression is a very early and transient phenomenon in all tested cells stimulated to proliferate [26, 57, 581. In continuously proliferating cells c-fos mRNA is barely detectable but remains inducible [59]. Activation of cfos gene expression seems to play a role in the control of cell proliferation, as antisense c-fos RNA reduces the growth of mouse tibroblasts in culture [60, 611. c-fos and Development
and Differentiation
High levels of c-fos transcripts are detected in extraembryonic (rapidly proliferating and bathed in a mixture of growth factors) tissues of mouse during development [33, 501. In embryonic tissues the highest levels of c-fos expression are observed in hemopoietic organs: fetal liver and bone marrow as well as in the skin. In adult tissues c-fos mRNA is easily detectable in some hemopoietic cells-bone marrow in general and in monocytes and neutrophils in particular [501. A rapid increase in c-fos mRNA levels follows induction of differentiation in numerous kinds of cells in vitro [26, 33, 501. The agents known to induce c-fos gene expression usually act via surface receptors and second messengers. On the contrary, differentiation of HL60 cells resulting from treatment with membranepenetrating DMSO does not lead to accumulation of c-fos mRNA [621. Cells whose induction of differentiation coincides with c-fos expression include teratocarcinomas, PC12 pheochromocytoma, and preadipocytes, where the rise in c-fos expression is extremely transient, and several hematopoietic cell lines, where cfos expression remains quite high, e.g., in cells differentiated into macrophages,
Molecular biology of cell activation
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or Il-3-dependent mast cell precursors [50]. The sustained high levels of c-fos mRNA in some hemopoietic cells suggests that in addition to being involved in cell activation, c-fos may have some cell type specific functions in the maintenance of a phenotype. As in the case of cell proliferation, the increased levels of c-fos mRNA during differentiation do not seem to be purely coincidental. In teratocarcinomas it has been shown that transfer of mouse or human c-fos protooncogenes provokes differentiation of F9 cells [63, 641. It is also conspicuous that during differentiation of preadipocytes into adipocytes the c-fos protein has been found in promoter region of genes selectively turned on by this process [53]. c-fos and Activation of Terminally Differentiated Cells The rapid and transient accumulation of c-fos mRNA is not limited to cellular proliferation and differentiation. Quite the opposite is true. c-fos gene expression seems to be a ubiquitous reaction of a great variety of cells to diversified stimuli like heat shock [65] or uv light [33]. An increased level of c-fos gene expression follows activation of macrophages with LPS [9] and stimulation of neurons with proconvulsants and epileptogenic treatments [66-68]. c-fos expression also has been implicated in physiological neuronal stimulation [69, 701 and found to be evoked by specific neurotransmitters [71-731. As yet, no data on the possible functional involvement of c-fos in the aforementioned phenomena are available. IV. ORNITHINE
DECARBOXYLASE
Omithine decarboxylase (ODC) catalyzes the conversion of ornithine to the diamine putrescine, a pivotal step in the biosynthesis of the polyamines spermidine and spermine [74, 751. ODC is the initial and rate-limiting enzyme in polyamine biosynthesis [74, 761. The ODC proteins localized predominantly in the cytoplasm, although spermine-the final product of polyamine biosynthesis-is found mainly in the nucleus [74, 771. Functional studies on omithine decarboxylase have benefited greatly from an application of its specific inhibitors. Difluoromethylomithine (DFMO) has proven to be particularly useful in this regard [74, 761. ODC and Proliferation An association of proliferation with a stimulation of polyamine biosynthesis has been repeatedly reported [74, 75, 781. The increase in polyamine content is believed to be a consequence of increased ODC activity. For example, a marked increase in the ODC activity occurs in regenerating rat liver during the first few hours after partial hepatectomy [79]; serum and growth factors like PDGF, EGF, and FGF induce ODC in human and rodent fibroblasts [80, 811; mitogenic lectins PHA and Con A stimulate ODC in human and mouse lymphocytes [82]. In different types of cells several peaks of ODC activity have been reported following cell cycle stimulation. The most pronounced of those peaks is detected at the mid to late Ga transition, and it has been shown to be dependent on the
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ODC mRNA accumulation. The increase in the ODC mRNA levels has been found following stimulation of the cell cycle of human and mouse fibroblasts and human T lymphocytes as well as mouse muscle cells [26, 83, 841. Similarly, as in the case of c-myc and c-&s, activation of the ODC gene expression can be brought about by competence growth factors. In mouse 3T3 fibroblasts PDGF (competence factor) is the strongest inducer of ODC gene expression [Sl], while EGF (progression factor) has a weaker potency. In contrast, in human WI38 fibroblasts EGF (in this case acting rather as a competence-like factor) is the only component of the serum-free medium able to cause ODC mRNA accumulation [83]. Similar to c-myc, the ODC mRNA levels are expressed invariantly in continuously proliferating cells 1831. The omithine decarboxylase enzymatic activity seems to be indispensable in order for cells to proliferate. The importance of ODC for cell proliferation is clearly illustrated by mutant P22 of Chinese hamster ovary cells which has no detectable ODC enzymatic activity [85]. In effect the cells cannot grow in the absence of exogenously added polyamines. Similarly, inhibition of the ODC arrests growing cells [75, 761. The exact nature of the ODC-dependent part of the cell cycle has not been clearly defined. It has been suggested that polyamines are crucial for the S phase [78], but recent studies point to the involvement of polyamines also in the activation of cell proliferation by competence growth factors like PDGF [86, 871. These findings are in good agreement with the gene expression data indicating a role of the competence growth factors in the regulation of the ODC mRNA levels (see above). ODC and Development
and Differentiation
There is compelling evidence that polyamines and ornithine decarboxylase play a role in the differentiation of mammalian cells. NGF, which induces the differentiation of PC12 cells, stimulates ODC gene expression within a few hours following treatment [SS]. Similarly differentiation of HL60 cells coincides with ODC mRNA accumulation (P. Aller, personal communication). The erythroid differentiation of Friend erythroleukemia cells is accompanied by an increase in ODC activity, which is protein biosynthesis dependent [89]. Moreover, DFMO can block this differentiation except when exogenous polyamines are provided [90]. Similarly, differentiation of L6 myoblasts and conversion of 3T3-Ll preadipocytes to adipocytes as well as differentiation of murine Friend erythroleukemia cells can be blocked by DFMO [78, 91-931. Polyamines also participate in critical developmental events during invertebrate and vertebrate embryogenesis. They play a fundamental role in gastrulation [78] and they are also probably involved in other processes as well, e.g., development of the nervous system [94]. ODC Induction
in Terminally
Differentiated
Cells
Stimuli which provoke the function of terminally differentiated cells may also lead to a rapid induction of the ODC. In rat liver an increase in ODC activity which is protein synthesis dependent has been observed following hypertonic
Molecular
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infusions, celite (a chemically inert mechanical irritant) injections, and other stressful procedures [79, 951. High levels of ornithine decarboxylase activity have been repeatedly observed in tissues actively involved in protein synthesis such as prostate [96] and in organs undergoing hypertrophy [97]. In androgen-induced hypertrophic growth of kidney, an extremely high increase in enzyme activity is preceded by ODC mRNA accumulation [98]. A similar induction of ODC occurs also in cardiac hypertrophy brought about by thyroxin treatment [94]. Stimulation of ODC activity has been also shown in hippocampal neurons after epileptogenie stimuli [99]. In LPS-treated mouse macrophages there is an accumulation of ODC mRNA (Kaczmarek et al., in preparation). V. CONCLUDING
REMARKS
The similarities among the genes described in this paper are briefly reviewed below : (1) All of the genes are activated by factors able to induce a new phenotype and, in particular, are responsible for an early phase of transition to a new phenotype (see also Ref. [loo]). (2) All of the genes are activated by external ligands acting via surface receptors and second messengers, particularly of phosphoinositide provenance. (3) The expression of all of the genes constitutes a primary response to external ligands; i.e., it is independent of the biosynthesis of other proteins induced by the ligands . (4) All of the mRNAs accumulate early after treatment with inductive stimuli and usually their level is only transiently increased, although it can stay persistently high in certain types of cells. (5) The protein products of all the genes have been shown to play a regulatory role in different forms of cell activation. (6) Mechanisms of expression of all of the gene are extremely complex, suggesting the necessity for the tine regulation of their cognate protein biosynthesis. We must emphasize clearly that we presume, on the merit of the largest amount of available data, that the c-myc, c-fos, and ODC are only selected examples of activation-related genes. Several studies suggest that there are additional (maybe even many) genes with similar characteristics. For example, it is interesting that the genes encoding cytoskeleton proteins (vimentin, actin, tubulin) have been found to belong to the genes that respond most rapidly to growth factors stimulating either proliferation or differentiation [26, 101, 1021.A similar pattern of expression has been also noted for protooncogenes other than c-myc and c-fos [26, 1031,such as a zinc finger encoding gene [104] as well as still undefined cell cycle-dependent genes obtained after differential screening of cDNA libraries [ 105-1071. We must also emphasize that by no means do we suggest that the genes described herein are an obligatory component of all the possible kinds of cellular 3-898337
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stimulation leading to the formation of a new phenotype. Similarly, we do not suggest that all three genes described in this paper must always be induced concomitantly. Instead we propose that the same genes can be involved in numerous seemingly unrelated, or even contradictory, phenomena. At this point the question of how so many different biological phenomena could be regulated by the same limited number of intracellular signals may arise. We suggest two, possibly complementary, explanations, First, most if not all of the phenomena described here of long-term phenotypic change are dependent on the synergistic action of more than one extracellular ligand. Most probably, the cooperation between these ligands drives the cell to a final response. Second, we suggest that the products of activation-related genes act on cells already predisposed to understand the signal delivered by these products. Let us assume that protein or enzymatic products of the activation-related genes regulate gene expression via interference with a chromatin structure of effector genes. It is possible that the chromatin structure of effector genes differs among different kinds of cells. For example, the genes encoding cytokines can be in “semi-open” chromatin conformation in dormant macrophages, while the genes encoding DNA synthesizing machinery are in “semi-open” chromatin structure in quiescent flbroblasts. In contrast, the latter genes have a “closed” chromatin conformation in macrophages and the former genes are in the same state of chromatin in fibroblasts. The “semi-open” chromatin is not expressed in the same manner as “closed” chromatin. However, the “semi-open” chromatin could be induced to gene expression by contact with products of activation-related genes like c-fos, cmyc, and ODC, appearing in cells treated with LPS in the case of macrophages or competent growth factors in the case of fibroblasts. In conclusion, our final thesis is that there is a ubiquitous genetic correlate of cellular activation, and the possible function of genes like c-myc, c-fos, and ODC seems therefore to be related to cellular activation per se rather than to stimulation of a given biological process. Because all of the proteins described in this review have been implicated in the direct or indirect regulation of gene expression, it is tempting to speculate that the described genes act as third messengers spreading within the nucleus an activation signal first delivered at the cell membrane and carried next by the second messengers.
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Nishikura, K., and Murray, J. (1987) Mol. Cell. Biol. 7, 639. Mitchell, R. C., Zokas, L., Schreiber, R. D., and Verma, I. M. (1985) Cell 40, 209. Muller, R., and Wagner, E. F. (1984) Nature (London) 311, 438. Ruther, U., Wagner, E. F., and Muller, R. (1985) EMBO .Z. 4, 1775. Andrews, G. K., Harding, M. A., Calvet, J. P., and Adamson, E. D. (1987) Mol. Cell. Biol. 7, 3452. Morgan, J. J., Cohen, D. R., Hempstead, J. L., and Curran, T. (1987) Science 237, 192. Dragunow, M., and Robertson, H. A. (1987) Neurosci. Lett. 82, 157. Kaczmarek, L., Siedlecki, J., & Danysz, W. (1988) Mol. Brain Res. 3, 183. Hunt, S. P., pini, A., and Evan, G. (1987) Nature (London) 328, 632. Curran, T., and Morgan, J. I. (1987) BioEssqs 7, 255. Szekely, A. M., Barbacia, M. L., and Costa, E. (1987) Neuropharmacology 26, 1779. Greenberg, M. E., Zii, E. B., and Greene, L. A. (1986) Science 234, 80. Barka, T., Gubits, R. M., and van der Noen, H. M. (1986) Mol. Cell. Biol. 6, 2984. Pegg, A. E. (1986) Biochem. J. 234,249. Tabor, C. W., and Tabor, H. (1984) Annu. Rev. Biochem. 53,749. Heby, 0. (1985) Adv. Enzyme Reg. 24, 103. Grille, R., and Fossa, T. (1982) Znt. .Z. Biochem. 15, 139. Heby, 0. (1981) DifSerentiution 19, 1. Schrock, T. R., Oakman, N. J., and Bucher, N. L. R. (1970) Biochim. Biophys. Acta 284,564. Stastry, M., and Cohen, S. (1970) Biochim. Biophys. Acfa 284. Hovis, J. G., Stumpo, D. J., Halsey, D. L., and Blackshear, P. J. (1986) .Z. Biol. Chem. 261, 10,380.
82. Bachrach, U., Menashe, M., Faber, J., Desser, H., and Seiler, N. (1981)Adv. Polyamine Res. 3, 259. 83. Kaczmarek, L., Calabretta, B., Ferrari, S., and de Riel, J. K. (1987) J. Cell. Physiol. 132, 545. 84. Olsson, E. N., and Spizz, G. (1986) Mol. Cell. Biol. 6, 2792. 85. Pohjanpelto, P., Holtta, E., and Janne, 0. A. (1985) Mol. Cell. Biol. 5, 1385. 86. Stimac, E., and Morris, D. R. (1987) .Z. Cell. Physiol. 133, 590. 87. Schefer, E. L., and Seidenfeld, J. (1987) .Z. Cell. Physiol. 133, 546. 88. Feinstein, S. C., Dana, S. L., McConlogue, L., Shooter, E. M., and Coffino, P. (1985) Proc. Natl. Acad. Sci. USA 82, 5761. 89. Gazitt, Y., and Friend, C. (1980) Cancer Res. 40, 1727. 90. Klinken, P. S., Billelo, J., Bauer, S., Morse, H. C., III., and Thorgeirsson, S. S. (1987) Cancer Res. 47, 2638.
91. Amri, E.-Z., Dani, C., Doglio, A., Etienne, J., Grimaldi, P., and Ailhaud, G. (1986) Biochem. J. 238, 115. 92. Watanabe, T., Sherman, M., Shafman, T., Iwata, T., and Kufe, D. (1986) J. Cell. Physiol. 127, 480.
93. Schindler, J., Kelly, M., and McCann, 9. P. (1985) J. Cell. Physiol. 122, 1. 94. Slotkin, T. A., and Bartolome, J. (1986) Brain Res. Bull. 17, 307. 95. Perry, J. W., and Oka, T. (1980) Biochim. Biophys. Acta 629, 24. %. Pegg, A. E., Lockwood, D. H., and Williams-Ashman, H. G. (1970) Biochem. J. 117, 17. 97. Janne, J., Poso, H., and Raina, A. (1978) Biochim. Biophys. Acta 473, 241. 98. Berger, F. G., Szymanski, P., Read, E., and Watson, G. (1984) J. Biol. Chem. 259, 7941. 99. Baudry, M., Lynch, G., and Gall, C. (1986) .Z. Neurosci. 6, 3430. 100. Baserga, R., and Fermi-i, S. (1987) Haematologica 72, 1. 101. Ferrari, S., Battini, R., Kaczmarek, L., Rittling, S., Calabretta, B., de Riel, K. J., Phiiponis, V., and Wei, J. F., Baserga, R. (1986) Mol. Cell. Biol. 6, 3614. 102. Mizobuchi, T., Yagi, Y., Mizuno, A., Matsuzaki, H., and Matsuda, M. (1988) Neurosci. Lett. 86,144.
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103. Muller, R. (1986) Trends Biochem. Sci. 11, 129. 104. Lau, L. F., andNathans, D. (1985) EMBOJ. 4, 3145. 105. Rollins, B. J., Morrison, E. D., and Stiles, C. D. (1987) Science 238, 1269. 106. Sukhatme, V. P., Cao, X., Chang, L. C., Tsai-Morris, C.-H., Stamenkovich, D., Ferreira, P. C. P., Cohen, D. R., Edwards, S. A., Shows, T. B., Curran, T., Le Beau, M. M., and Adamson, E. D. (1988) Cell 53, 37. 107. Almerdal, J. M., Sommer, D., MacDonald-Bravo, H., Burckhardt, J., Perera, J., and Bravo, R. (1988) Mol. Cell. Biol. 8, 2140. Received August 30, 1988 Revised version received December 12, 1988
SPECIAL ARTICLE Molecular
Biology
LESZEK KACZMAREK**’ Departments
of *Neurophysiology Experimental Biology,
of Cell Activation and BOZENA KAMINSKA?
and TCellular Biochemistry, Nencki Institute Pasteura 3, 02-093 Warsaw, Poland
of
This article summarizes common features of activation of diierent types of cells along diierent physiological lines such as proliferation, differentiation, and execution of function of terminally differentiated cells. The common basis of many of these phenomena includes (i) first messengers (growth factors, cytokines, neurotransmitters, etc.) acting on membrane receptors, (ii) second messengers (CAMP, IPr, DAG, Ca2’) spreading an activating signal inside the cell, and (iii) elevated expression of some genes (c-fos, c-myc, omithine decarboxylase). The role of the genetic correlate in cell activation is emphasized, and it is concluded that the aforementioned genes (their protein products) should be called third messengers, whose function is mediation of long-term phenotypic changes. @ 1989 Acadetmc Press, Inc.
I. ACTIVATION
VERSUS MAINTENANCE
OF A PHENOTYPE
The term “cell activation” can be used to describe a plethora of different biological phenomena, some of which are seemingly unrelated. In this paper we suggest that the same term can designate an early phase of (predisposal to?) biological processes which have in common a long-term phenotypic change, e.g., stimulation of quiescent cells to enter the cell cycle, induction of differentiation, and long-lasting functional activity of terminally differentiated cells such as macrophages or neurones. At the beginning of our discussion we must distinguish in all of the phenomena the activation of a new phenotype from the maintenance of this phenotype. We propose that cell activation is a ubiquitous phenomenon preceding and leading to establishment of a new phenotype. In order to enter the cell cycle quiescent (Go) cells require treatment with growth factors [l]. For cell cycle stimulation normal, nontransformed cells require more than one growth factor. The best characterized cells in this regard, mouse Balb/c 3T3 fibroblasts, may serve as a model of mechanisms controlling cell proliferation [2]. There are two groups of growth factors acting in concert on those cells. Competence factors [e.g., PDGF (platelet-derived growth factor)] initiate some biochemical reactions in quiescent Balb/c 3T3 fibroblasts and in this way they prime the cells to be responsive to progression factors [EGF (epidermal growth factor) and IGF-I (insulin like growth factor I) are the most prominent examples]. Cells treated with factors belonging to both groups enter DNA synthe-
’ To whom reprint requests should be addressed. Copyright @$J1969 by Academic Press, Inc. All rights of reproductmn m any form reserved 0014-4827189 $03 00
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Molecular
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25
sis. Neither competence nor progression factors alone (at certain concentrations) suffice to stimulate the G,,-S transition of Balb/c 3T3 cells. Interestingly, the competence phase of the G& transition is present for a long time after withdrawal of the competence factors-the cells “remember” the factors they were treated with [3, 41. This means that factors responsible for the activation of the G&S transition are not required for maintenance of the active state. In cellular differentiation it is possible to clearly distinguish the induction of the terminal phenotype from its maintenance, and as in the cell cycle, differentiation inducers are very often not required to be permanently present during the transition to a new phenotype, For example, a 5-min treatment of HL60 human promyelocytic leukemia cells with phorbol esters (TPA) provokes differentiation of the cells into macrophages [5]. Similarly, differentiation of chicken myoblasts into myotubes resulting from the treatment of insulin requires only 1 min of contact between the hormone and the cells [6]. A similar phenomenon can also be observed in terminally differentiated cells. For example, neurons can be primed to increased activity by electrical stimulation, known as kindling. The enhanced neuronal excitability observed in this epilepsy-like phenomenon can still be detected well after the stimulation has been discontinued [7, 81. Also inflammation-mimicking activation of macrophages with lipopolysaccharide (LPS) drives the cells to produce cytokines even after the LPS has been removed [9]. Interestingly, all of the aforementioned phenomena of cell activation follow a similar general pathway. First, the external stimuli (first messengers) activate their specific receptors. Next, this signal is enhanced and spread inside the cells with the aid of second messengers. Finally, the specific effector systems are set up. The first messengers mentioned here (neurotransmitters, growth factors, etc.) are hydrophilic and do not easily penetrate the cell membrane. They act via their specific surface receptors and formation of second messengers. The first messengers can produce long-lasting effects often going beyond the limits of the life span of the first messenger itself. Nevertheless, the formation of the second messengers cannot explain the long duration of the signal. All of the known second messenger systems result in the activation of specific protein kinases (CAMP-dependent kinase A, Ca’+-driven kinases dependent on calmodulin, diacylglycerol-dependent kinase C, etc.) [IO-131. Therefore, their biologial effects last within the limits of the persistence of protein phosphorylation, which very often is short-lived due to a fine regulation by phosphatases [14]. However, the effects of external activation can last even for days and weeks after the stimulus application. It is widely accepted that this phenomenon can be explained by an indirect effect of first messengers on gene expression leading to the elevated production of specific mRNAs and proteins. These proteins performing their physiological role (enzymatic, structural, etc.) can in turn modify the phenotype of the cell for quite a long time. We propose that the genes encoding proteins constituting the new phenotype be designated “effector genes.” In this paper we reivew data suggesting that it is possible to distinguish also
26
Kaczmarek and Kamihka
another class of genes, whose expression is dependent on external signals-“activation-related’genes.” While the effector genes are usually specific for the given kind of cell stimulation, the activation-related genes are at least partially expressed ubiquitously in different types of cells stimulated by a variety of biological phenomena. Moreover, their function seems to be related more to the activation than to the maintenance of a phenotype. We have selected three genes, which in our opinion best exemplify our thesis about the existence of this class of genes. II. c-myc c-myc is a cellular counterpart of the transforming gene (v-myc) of MC29, OKlO, CMII, MH2, myc-FeLV leukemic viruses [15-171. It has been detected in the genomes of all vertebrates tested so far [18]. The v-myc (viral) and c-myc (cellular) proteins are found in cell nuclei [15, 193,which leads to the suggestion that they are’ somehow involved in nuclear functions, e.g., control of gene expression. In fact, it has been shown that both v-myc and c-myc can act as transcription activators [20, 211. The c-myc protein has been found to be DNAbinding and localized to the chromatin and detected even more abundantly in the nuclear matrix 1221.On the other hand, no sequence specificity of binding of cmyc protein to DNA has been established. There is also experimental evidence for the thesis that the protein may be involved in another step in the regulation of gene expression, namely post-transcriptional processing of RNA [23]. c-myc and Cell Proliferation The most obvious support for the theory of c-myc involvement in the control of cell proliferation comes from the fact that v-myc is an oncogene. Moreover, cmyc driven by tissue-specific promoters and enhancers induces malignancies in target organs in transgenic mice [24, 253. This means that the myc gene can provide cells with a phenotype manifested by abnormal proliferation. The involvement of c-myc in normal cellular proliferation is supported by data on the pattern of its expression. The accumulation of c-myc mRNA has been observed in all kinds of Go cells stimulated to proliferate (for review see [26]). The pattern of c-myc mRNA levels always follows similar kinetics. There is an early increase, beginning at one to a few hours after application of the stimulus, the maximal levels of c-myc mRNA are reached in mid to late G& transition, and then they decline. In the second and following cell cycles the mRNA levels of cmyc are signiticantly lower than those during the G& transition and are invariant throughout the phases of the cell cycle of proliferating cells 1271. In numerous cases the factors stimulating c-myc expression have been identitied. In general, they are competence or competence-like factors, i.e., factors responsible for the early phase of the G,,-S transition. All of them act via surface receptors and second messengers. There is much information suggesting that the increase in c-myc mRNA (and protein) levels following cell cycle stimulation is not purely coincidental. Introduction of the c-myc coding sequences under the control of inducible or constitu-
Molecular
biology of cell activation
27
tive promoters into mouse fibroblasts decreases the dependence of those cells on competence growth factors [28]. Similarly, microinjection of recombinant c-myc protein renders tibroblasts competent [29]. Introduction of highly expressable vmyc carrying constructs into hematopoietic/lymphoid cell lines also abrogates their dependence on growth factors [30]. Moreover, depletion of c-myc mRNA (and protein) resulting from c-myc antisense oligonucleotide treatment inhibits transition from Go to S of human T lymphocytes [31]. c-myc and Cellular Differentiation
A brief examination of c-myc expresson in differentiating cells further suggests a close link between c-myc and cell proliferation. During hemopoietic differentiation of leukemic cell lines [HL60, Friend murine erythroleukemia (F-MEL)], as well as during differentiation of teratocarcinomas and myoblasts in vitro, the decrease of c-myc expression was observed concomitantly with the declining proliferation rate of the cells [16, 32, 331. Moreover, introduction of c-myc or vmyc under constitutive promoters into F-MEL or myoblasts prevented or delayed differentiation of these cells following treatment with appropriate stimuli [34-361.
On the other hand, a closer look at the kinetics of c-myc expression in the aforementioned cells raises questions about the simple notion just described. There is indeed a dramatic decrease of c-myc mRNA levels in cells stimulated to differentiate; however, in F-MEL cells the decrease is followed by a transient rise in c-myc expression preceding terminal differentiation [37-40]. A similar phenomenon can also be observed during myogenesis [41] and differentiation of murine embryonal carcinoma cells [42]. Unfortunately, the data which present sufficiently long and detailed kinetics of c-myc mRNA levels in other cell lines are lacking. The early decrease in c-myc mRNA levels following induction of differentiation can easily be explained by the concomitant cessation of cell proliferation, as we have mentioned that c-myc is expressed, albeit at a low level, in growing cells. We interpret the following rise in c-myc expression as indicative of its involvement in the activation of differentiation. Incidentally, Caffrey et al. [36], who extensively examined differentiation of B&H1 myoblasts, observed a delay rather than an inhibition of the process following introduction of exogenous cmyc into the cells. Finally, it is noteworthy that the often-studied HL60 cells may be a particularly difficult model with which to investigate c-myc involvement in cellular differentiation. The high level of amplification of the c-myc gene in these cells [ 171probably leads to a change in the normal regulation of gene expression; hence, it is difficult to extend data obtained with this cell line to cells with normal c-myc regulation. Moreover, there are reports showing an increase in c-myc expression in cells stimulated to differentiate concomitant with the cessation of proliferation. The best-known example is rat pheochromocytoma PC12 cells treated with nerve growth factor (NGF) [43, 441. Correlation of augmented c-myc expression and differentiation has been also suggested for Purkinje cells in mouse cerebellum r451.
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In developing human embryos only a partial correlation between proliferation and c-myc expression has been noted [46-48], indicating again that not only proliferating cells are a target for c-myc biological activities. Similarly in early embryonic development of Xenopus oocytes it was difficult to correlate c-myc mRNA accumulation exclusively with cell proliferation [49]. c-myc in Terminally
Differentiated
Cells
In mouse peritoneal macrophages an increase of c-myc expression has been found following treatment with LPS, known to be an inducer of specialized inflammation-related functions of macrophages [9]. III. C-j-OS c-fos is a normal cellular counterpart of FBJ and FBR osteosarcoma transforming gene (v-fos) [26, 501. c-fos, like c-myc, is a nuclear protein [19, 501 and it has been localized to the chromatin [51, 521, where it can bind specific regions of DNA 1531,which strongly suggests its role as a gene expression regulator. In fact, both v-fos and c-fos have been shown to be able to regulate transcription [54-561. c-fos and Cell Proliferation
Again, as with c-myc, before reviewing data describing the c-fos role in the cell cycle, we must stress that it is a homolog of an oncogene. Activation of c-fos expression is a very early and transient phenomenon in all tested cells stimulated to proliferate [26, 57, 581. In continuously proliferating cells c-fos mRNA is barely detectable but remains inducible [59]. Activation of cfos gene expression seems to play a role in the control of cell proliferation, as antisense c-fos RNA reduces the growth of mouse tibroblasts in culture [60, 611. c-fos and Development
and Differentiation
High levels of c-fos transcripts are detected in extraembryonic (rapidly proliferating and bathed in a mixture of growth factors) tissues of mouse during development [33, 501. In embryonic tissues the highest levels of c-fos expression are observed in hemopoietic organs: fetal liver and bone marrow as well as in the skin. In adult tissues c-fos mRNA is easily detectable in some hemopoietic cells-bone marrow in general and in monocytes and neutrophils in particular [501. A rapid increase in c-fos mRNA levels follows induction of differentiation in numerous kinds of cells in vitro [26, 33, 501. The agents known to induce c-fos gene expression usually act via surface receptors and second messengers. On the contrary, differentiation of HL60 cells resulting from treatment with membranepenetrating DMSO does not lead to accumulation of c-fos mRNA [621. Cells whose induction of differentiation coincides with c-fos expression include teratocarcinomas, PC12 pheochromocytoma, and preadipocytes, where the rise in c-fos expression is extremely transient, and several hematopoietic cell lines, where cfos expression remains quite high, e.g., in cells differentiated into macrophages,
Molecular biology of cell activation
29
or Il-3-dependent mast cell precursors [50]. The sustained high levels of c-fos mRNA in some hemopoietic cells suggests that in addition to being involved in cell activation, c-fos may have some cell type specific functions in the maintenance of a phenotype. As in the case of cell proliferation, the increased levels of c-fos mRNA during differentiation do not seem to be purely coincidental. In teratocarcinomas it has been shown that transfer of mouse or human c-fos protooncogenes provokes differentiation of F9 cells [63, 641. It is also conspicuous that during differentiation of preadipocytes into adipocytes the c-fos protein has been found in promoter region of genes selectively turned on by this process [53]. c-fos and Activation of Terminally Differentiated Cells The rapid and transient accumulation of c-fos mRNA is not limited to cellular proliferation and differentiation. Quite the opposite is true. c-fos gene expression seems to be a ubiquitous reaction of a great variety of cells to diversified stimuli like heat shock [65] or uv light [33]. An increased level of c-fos gene expression follows activation of macrophages with LPS [9] and stimulation of neurons with proconvulsants and epileptogenic treatments [66-68]. c-fos expression also has been implicated in physiological neuronal stimulation [69, 701 and found to be evoked by specific neurotransmitters [71-731. As yet, no data on the possible functional involvement of c-fos in the aforementioned phenomena are available. IV. ORNITHINE
DECARBOXYLASE
Omithine decarboxylase (ODC) catalyzes the conversion of ornithine to the diamine putrescine, a pivotal step in the biosynthesis of the polyamines spermidine and spermine [74, 751. ODC is the initial and rate-limiting enzyme in polyamine biosynthesis [74, 761. The ODC proteins localized predominantly in the cytoplasm, although spermine-the final product of polyamine biosynthesis-is found mainly in the nucleus [74, 771. Functional studies on omithine decarboxylase have benefited greatly from an application of its specific inhibitors. Difluoromethylomithine (DFMO) has proven to be particularly useful in this regard [74, 761. ODC and Proliferation An association of proliferation with a stimulation of polyamine biosynthesis has been repeatedly reported [74, 75, 781. The increase in polyamine content is believed to be a consequence of increased ODC activity. For example, a marked increase in the ODC activity occurs in regenerating rat liver during the first few hours after partial hepatectomy [79]; serum and growth factors like PDGF, EGF, and FGF induce ODC in human and rodent fibroblasts [80, 811; mitogenic lectins PHA and Con A stimulate ODC in human and mouse lymphocytes [82]. In different types of cells several peaks of ODC activity have been reported following cell cycle stimulation. The most pronounced of those peaks is detected at the mid to late Ga transition, and it has been shown to be dependent on the
30
Kaczmarek and Kamihka
ODC mRNA accumulation. The increase in the ODC mRNA levels has been found following stimulation of the cell cycle of human and mouse fibroblasts and human T lymphocytes as well as mouse muscle cells [26, 83, 841. Similarly, as in the case of c-myc and c-&s, activation of the ODC gene expression can be brought about by competence growth factors. In mouse 3T3 fibroblasts PDGF (competence factor) is the strongest inducer of ODC gene expression [Sl], while EGF (progression factor) has a weaker potency. In contrast, in human WI38 fibroblasts EGF (in this case acting rather as a competence-like factor) is the only component of the serum-free medium able to cause ODC mRNA accumulation [83]. Similar to c-myc, the ODC mRNA levels are expressed invariantly in continuously proliferating cells 1831. The omithine decarboxylase enzymatic activity seems to be indispensable in order for cells to proliferate. The importance of ODC for cell proliferation is clearly illustrated by mutant P22 of Chinese hamster ovary cells which has no detectable ODC enzymatic activity [85]. In effect the cells cannot grow in the absence of exogenously added polyamines. Similarly, inhibition of the ODC arrests growing cells [75, 761. The exact nature of the ODC-dependent part of the cell cycle has not been clearly defined. It has been suggested that polyamines are crucial for the S phase [78], but recent studies point to the involvement of polyamines also in the activation of cell proliferation by competence growth factors like PDGF [86, 871. These findings are in good agreement with the gene expression data indicating a role of the competence growth factors in the regulation of the ODC mRNA levels (see above). ODC and Development
and Differentiation
There is compelling evidence that polyamines and ornithine decarboxylase play a role in the differentiation of mammalian cells. NGF, which induces the differentiation of PC12 cells, stimulates ODC gene expression within a few hours following treatment [SS]. Similarly differentiation of HL60 cells coincides with ODC mRNA accumulation (P. Aller, personal communication). The erythroid differentiation of Friend erythroleukemia cells is accompanied by an increase in ODC activity, which is protein biosynthesis dependent [89]. Moreover, DFMO can block this differentiation except when exogenous polyamines are provided [90]. Similarly, differentiation of L6 myoblasts and conversion of 3T3-Ll preadipocytes to adipocytes as well as differentiation of murine Friend erythroleukemia cells can be blocked by DFMO [78, 91-931. Polyamines also participate in critical developmental events during invertebrate and vertebrate embryogenesis. They play a fundamental role in gastrulation [78] and they are also probably involved in other processes as well, e.g., development of the nervous system [94]. ODC Induction
in Terminally
Differentiated
Cells
Stimuli which provoke the function of terminally differentiated cells may also lead to a rapid induction of the ODC. In rat liver an increase in ODC activity which is protein synthesis dependent has been observed following hypertonic
Molecular
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31
infusions, celite (a chemically inert mechanical irritant) injections, and other stressful procedures [79, 951. High levels of ornithine decarboxylase activity have been repeatedly observed in tissues actively involved in protein synthesis such as prostate [96] and in organs undergoing hypertrophy [97]. In androgen-induced hypertrophic growth of kidney, an extremely high increase in enzyme activity is preceded by ODC mRNA accumulation [98]. A similar induction of ODC occurs also in cardiac hypertrophy brought about by thyroxin treatment [94]. Stimulation of ODC activity has been also shown in hippocampal neurons after epileptogenie stimuli [99]. In LPS-treated mouse macrophages there is an accumulation of ODC mRNA (Kaczmarek et al., in preparation). V. CONCLUDING
REMARKS
The similarities among the genes described in this paper are briefly reviewed below : (1) All of the genes are activated by factors able to induce a new phenotype and, in particular, are responsible for an early phase of transition to a new phenotype (see also Ref. [loo]). (2) All of the genes are activated by external ligands acting via surface receptors and second messengers, particularly of phosphoinositide provenance. (3) The expression of all of the genes constitutes a primary response to external ligands; i.e., it is independent of the biosynthesis of other proteins induced by the ligands . (4) All of the mRNAs accumulate early after treatment with inductive stimuli and usually their level is only transiently increased, although it can stay persistently high in certain types of cells. (5) The protein products of all the genes have been shown to play a regulatory role in different forms of cell activation. (6) Mechanisms of expression of all of the gene are extremely complex, suggesting the necessity for the tine regulation of their cognate protein biosynthesis. We must emphasize clearly that we presume, on the merit of the largest amount of available data, that the c-myc, c-fos, and ODC are only selected examples of activation-related genes. Several studies suggest that there are additional (maybe even many) genes with similar characteristics. For example, it is interesting that the genes encoding cytoskeleton proteins (vimentin, actin, tubulin) have been found to belong to the genes that respond most rapidly to growth factors stimulating either proliferation or differentiation [26, 101, 1021.A similar pattern of expression has been also noted for protooncogenes other than c-myc and c-fos [26, 1031,such as a zinc finger encoding gene [104] as well as still undefined cell cycle-dependent genes obtained after differential screening of cDNA libraries [ 105-1071. We must also emphasize that by no means do we suggest that the genes described herein are an obligatory component of all the possible kinds of cellular 3-898337
32
Kaczmarek and Kamiriska
stimulation leading to the formation of a new phenotype. Similarly, we do not suggest that all three genes described in this paper must always be induced concomitantly. Instead we propose that the same genes can be involved in numerous seemingly unrelated, or even contradictory, phenomena. At this point the question of how so many different biological phenomena could be regulated by the same limited number of intracellular signals may arise. We suggest two, possibly complementary, explanations, First, most if not all of the phenomena described here of long-term phenotypic change are dependent on the synergistic action of more than one extracellular ligand. Most probably, the cooperation between these ligands drives the cell to a final response. Second, we suggest that the products of activation-related genes act on cells already predisposed to understand the signal delivered by these products. Let us assume that protein or enzymatic products of the activation-related genes regulate gene expression via interference with a chromatin structure of effector genes. It is possible that the chromatin structure of effector genes differs among different kinds of cells. For example, the genes encoding cytokines can be in “semi-open” chromatin conformation in dormant macrophages, while the genes encoding DNA synthesizing machinery are in “semi-open” chromatin structure in quiescent flbroblasts. In contrast, the latter genes have a “closed” chromatin conformation in macrophages and the former genes are in the same state of chromatin in fibroblasts. The “semi-open” chromatin is not expressed in the same manner as “closed” chromatin. However, the “semi-open” chromatin could be induced to gene expression by contact with products of activation-related genes like c-fos, cmyc, and ODC, appearing in cells treated with LPS in the case of macrophages or competent growth factors in the case of fibroblasts. In conclusion, our final thesis is that there is a ubiquitous genetic correlate of cellular activation, and the possible function of genes like c-myc, c-fos, and ODC seems therefore to be related to cellular activation per se rather than to stimulation of a given biological process. Because all of the proteins described in this review have been implicated in the direct or indirect regulation of gene expression, it is tempting to speculate that the described genes act as third messengers spreading within the nucleus an activation signal first delivered at the cell membrane and carried next by the second messengers.
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