Are all nonproliferating cells similar?

ExperimentalGerontology,Vol. 27, pp. 419--423, 1992

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ARE ALL NONPROLIFERATING

CELLS SIMILAR?

EUGENIA WANG The Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, and Departments of Medicine and Anatomy, McGill University, Montr6al, Qu6bec, Canada Key Words: nonproliferating ceils, statin, terminin, DNA, kinase

INTRODUCTION THE ANSWERto this article's question is no; the obvious example to support this negative statement is the difference between quiescent and senescent cells. The most readily measurable phenotypic characteristic distinguishing these cells from their proliferating counterparts is the fact that the former group of cells cannot incorporate 3H-thymidine, which would indicate the presence of DNA synthesis (Hayflick, 1961; Dell'Orco et al., 1973). For quiescent cells, this phenomenon of not synthesizing DNA is transient, and can be restored once the blockage for growth, such as serum starvation, is removed. For senescent fibroblasts, the barrier to further growth is permanent; and except for a few experimental manipulations, such as introduction of simian virus 40 (SV40) T-antigen (Peacocke and Campisi, 1991), in vitro aged cells have lost their capability of DNA synthesis for good (Phillips et al., 1987; Goldstein, 1990). Therefore, quiescent and senescent fibroblasts can be easily distinguished by whether their experience of growth arrest is reversible or not. PRODUCTION OF STATIN AND TERMININ MONOCLONAL ANTIBODIES AS MARKERS FOR NONPROLIFERATING CELLS IN GENERAL, AND SENESCENT FIBROBLASTS IN PARTICULAR The research focus in our laboratory has been to identify gene expressions that are uniquely activated in nonproliferating cells. Several lines of strong evidence have clearly described that the cessation of proliferation in senescent fibroblasts is a dominant phenomenon, which may require the activation of specific genes to direct entry into the nongrowing state (Matsumura et aL, 1980; Rabinovitch and Norwood, 1980; Pereira-Smith and Smith, 1981; Stein and Yanishevsky, 1981; Lumpkin et aL, 1986). Based on this work, we hypothesized the existence of nonproliferation-specific gene expressions, whose protein products are present only in cells that cease to synthesize DNA. In this context, we Correspondence to: E. Wang, The Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, 3755, chemin Cfte Ste.-Catherine, Montreal, Quebec, Canada H3T 1E2. 419

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have used hybridoma technology to produce monoclonal antibodies identifying some of these proteins, and thus serving as biochemical markers to segregate nongrowing from growing cells. For example, we have produced an unique antibody, $44, which detects a protein, statin, only in nongrowing cell nuclei (Wang, 1985a,b; Wang and Krueger, 1985; Wang and Lin 1986; Wang, 1987; Wang, 1989; Ching and Wang, 1990). The successful production of statin antibody, however, does not allow us to further separate quiescent cells from their senescent counterparts. The need to advance our attempt in this direction prompted us to undertake the production of a monoclonal antibody recognizing only in vitro aged fibroblasts, by immunosuppression coupled with hybridoma technology (Wang and Tomaszewski, 1991); the result was the production ofmonoclonal antibody 1.2 (mab 1.2). Immunostaining characterization of the mab 1.2 antibody shows that terrninin is localized only in senescent fibroblasts, in a cytoplasmic granular pattern. Thus, by immunofluorescence microscopy, nongrowing cells can be distinguished from their growing counterparts by the presence of nuclear statin, and within this group those that are irreversibly growth arrested, such as senescent fibroblasts, can be separated from quiescent cells by the presence ofterminin-positive granules in the cytoplasm. The following paragraphs are a summary of our findings so far on the characterization of statin and terminin as a function of cessation of proliferation in both culture systems and tissues, and the biochemical and molecular investigation of these two proteins in terms of mechanisms for control of proliferation. Furthermore, the technology developed in studying these two proteins can be used as a model for studying other nonproliferationspecific gene activations. STATIN BIOCHEMISTRY, PURIFICATION, KINASE, ETC. To ease our task in purification of native statin, we decided to use statin-positive liver as our source of starting materials, rather than in vitro aged senescent fibroblast cultures. Native statin can be purified from rat liver through step-wise biochemical procedures involving ammonium sulfate precipitation, DEAE, heparin-sepharose, and column chromatography (Sester et aL, 1990). The purified protein band analyzed on two-dimensional gels shows two isomeric groupings, with isoelectric focusing points (pls) of 6.8 and 7.2. Further analysis of immunoprecipitation with phospho-labeled senescent fibroblasts shows that statin is phosphorylated. By detailed in situ assays of how statin becomes phosphorylated, we found an associated serine and theonine kinase with a molecular weight of 46 kilodaltons (kDa). This statin-associated kinase may prove to be of significant importance in the control of cell cycle traversal. We are currently testing whether similarities exist between statin-associated kinase and cdc2 kinase, and whether statin could serve as a sequestering element to the kinase needed for the cell cycle control gene or antioncogenes such as the retinoblastoma (Rb) gene. STATIN AS A NEGATIVE INDICATOR FOR TISSUE NEOPLASIA The fact that statin is found only in nonproliferating cells suggests the use of the nuclear presence of statin as a marker for the growth status of tissues. This approach is especially important in view of the fact that one cannot readily label living animals with 3H-thymidine to assay the level of DNA synthesis. Furthermore, in extracting clinical samples of human disease situations, frequently little or no preparatory procedure can be performed before the biopsy manipulation. Therefore, it is most significant that an in situ marker for

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growth is now available, in particular in tumor situations where evaluation of growth status is important for prognostic or diagnostic purposes. Based on this rationale, we have used statin's presence in immunohistochemistry assays of growth status of several human tumor situations such as breast carcinoma, colorectal carcinoma, and brain tumors; in all cases, the presence of statin is inversely related to the degree of the malignant states of the tumors. In particular, we have performed a comprehensive study with 52 brain tumors (Tsanaclis et al., 1991). Here, statin's presence is evaluated in parallel with a proliferationspecific marker, Ki67; our results show that the presence of statin is a more sensitive marker than Ki67, which can only differentiate metastatic and malignant cases, and fails to separate the latter from benign tumors. The use ofstatin as a negative marker for cell growth can also be coupled with the expression of oncogenes and antioncogenes. We have evaluated statin presence in colorectal carcinoma to address the question of how far away from the tumor normal cell growth status persists. Although nearer and farther sites from the samples seem morphologically identical, our results with anti-p53, anfi-c-fos, and anti-statin antibodies show that, at least up to 3 cm away from the tumor area, cells are negative for statin but remain positive for cfos and p53. This result confirms the suggestion that there is a field defect in tumor ontogeny, which can be determined by the presence of statin as a negative index. IDENTIFICATION OF TERMININ AS A MARKER FOR SENESCENT FIBROBLASTS ONLY While the presence of statin is useful in distinguishing growing cells from their nongrowing counterparts, this phenotypic characteristic cannot be used for separating senescent from quiescent fibroblasts. It is therefore our goal, following the development ofstatin antibody, to devise a means to generate a monoclonal antibody that labels only senescent fibroblasts. Toward this goal, we used immunosuppression to generate a monoclonal antibody (mab 1.2) that reacts only with cytoplasmic granules found in senescent fibroblasts (Wang and Tomaszewski, 1991). We call the protein recognized by mab 1.2 terminin, reflecting the property of its finality in the process of in vitro aging. Upon examining cultures at different in vitro life spans, we identified terminin presence in young cell cultures too, but at a much lower level. With an increase in the number of population doubling levels (PDLs), there is a direct increase in the number of termininpositive cells; this result confirms the early observation (Smith and Hayflick, 1974; Smith and Whitney, 1980) that colony heterogeneity exists in human fibroblast cultures, and that the senescent phenotype can even be found on a smaller scale in young cell cultures. Conversely, in senescent cultures, only 85% of cells show terminin positivity, reflecting the fact that a smaller proportion of the cell population does not acquire the typical old cell phenotype even at the end of their in vitro life span. BIOCHEMICAL CHARACTERIZATION OF TERMININ SHOWS STEPLADDER PROTEOLYSIS AS A POSTTRANSLATIONAL MODIFICATION FOR ITS SPECIFIC SENESCENCE-DEPENDENT PRESENCE Protein identification by Western blotting shows that, on sodium decyl sulfate (SDS) denatured gels, mab 1.2 recognizes a protein of 90 kDa in young growing and quiescent fibroblasts, but reacts with a protein of 60 kDa in senescent cells. This result suggests that the protein is present in all three cell types, but the staining reaction seen by immunoflu-

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orescence microscopy is the 60-kDa form. The discrepancy between the immunoblotting result and that from immunofluorescence is due to the fact that the former assays use denatured protein, where all antigenic epitopes are unfolded, and allows the reaction of mab 1.2 to proceed further, while the latter assay only recognizes the protein in its native form, with possible masking ofepitopes because of protein-folding structures. Nevertheless, this result demonstrates posttranslational modification via proteolysis as a key regulatory event for terminin's distinct identity in senescent cells. TERMININ PRESENCE AND P R O G R A M M E D CELL DEATH An accidental failure to feed a confluent culture of young fibroblasts opened a new way to examine the biology ofterminin expression. In a culture unfed for 3 weeks, a significant portion of the cell monolayer changes from negative to positive staining for mab 1.2 antibody. This result prompted us to evaluate the possibility that this change in staining reaction may be due to those cells on the way to death. To determine whether the process leading to death can indeed be reflected by positive immunostaining reaction to mab 1.2, we adapted the serum deprivation procedure in mouse 3T3 fibroblasts. Our results show that mab 1.2 staining can be observed in those cells that have been deprived of serum for 48 h; the cells are on their way to death, and by 91 h the majority of cells are dead. Immunoblotting results show that at 0 h the terminin polypeptide is predominantly in the form of 90 kDa, while at 24 h after removal of serum the predominant protein species is in the form of 60 kDa with a faint positive band at 30 kDa; by 48 h, the 30-kDa band is predominant. This appears to illustrate a ladder type of proteolysis from 90 kDa to 60 kDa, and then to 30 kDa. The final proteolytic product of 30 kDa is then the marker signaling the onset of death. Therefore, one may expect the presence of the 90-kDa form in young cells, whether growing or not; 60 kDa in irreversibly growth-arrested cells such as senescent fibroblasts; and the 30-kDa form in cells that experience the onset of programmed cell death. We have further examined patterns ofterminin presence in a well-established model of programmed cell death in tissues. One of these models is axotomy of the neuroretinal ganglion. In these neurons, if one severs the axons, a controlled program of death is initiated; unique gene expressions are required for this event to occur. Our results with this system show that indeed a positive staining reaction for mab 1.2 antibody is observed during the process of dying. Furthermore, we observe that in cultures of sympathetic ganglion, a model of the peripheral nervous system, removal of nerve growth factor (NGF) will initiate programmed cell death and also show positive staining for mab 1.2 antibody. Immunoblotting results also show that in those dying neurons terminin is present in the 30-kDa form, which therefore further supports the suggestion that the 30-kDa form is a marker for the onset of cell death. CONCLUSION AND SUMMARY The investigation of the biology of statin and terminin has provided us the means to identify different physiological stages of cells in relation to the life span of fibroblasts. To begin with, one can use the presence of these proteins as biochemical and histochemical markers for their growth status. Of much greater importance, these studies reveal to us two most essential biochemical events of posttranslational modification: kinase phosphorylation and protease proteolysis. From the statin-associated kinase study, one may begin to

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evaluate whether the selective activity of this kinase is essential to the maintenance of the nonproliferative state, and thereby begin to approach questions on the activation of statin expression as the cause or consequence of the arrest of cell proliferation. From the terminin study, one can not only use the three different molecular weight forms to distinguish senescent cells from young fibroblasts as well as to identify those cells that are dying, but also to understand how a key biochemical process such as proteolysis is related to events occurring in young, old, and dying states. REFERENCES CHING, G. and WANG, E. Characterization of two populations of statin and the relationship of their synthesis to the state of cell proliferation. J. Cell Biol. 1 lfl, 255-26 l, 1990. DELL'ORCO, R.T., MERTENS, J.G., and KRUSE, P.F. Doubling potential, calendar time, and donor age of human diploid cells in culture. Exp. CellRes. 77, 356-360, 1973. GOLDSTEIN, S. Replicative senescence: The human fibroblast comes of age. Science 249, I 129- l 133, 1990. HAYFLICK, L. The limited in vitro lifespan of human diploid cell strains. Exp. CellRes. 37, 614-636, 1965. LUMPKIN, C.K., JR., McCLUNG, J.K., PEREIRA-SM1TH, O.M., and SMITH, J.R. Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts. Science 232, 393-397, 1986. MATSUMURA, T., PFENDT, A., ZERRUDO, Z., and HAYFLICK, L. Senescent human diploid cells (W138) attempted induction of proliferation by injection with SV-40 and by fusion with irradiated continuous cell lines. Exp. CellRes. 125, 453-457, 1980. PEACOCKE, M. and CAMPISI, J. Cellular senescence: A reflection of normal growth control, differentiation, or aging? J. Cell. Biochem. 45, 147-155, 1991. PEREIRA-SM1TH, O.M. and SMITH, J.R. Expression of SV-40 T antigen in finite lifespan hybrids of normal and SV-40-transformed fibroblasts. Somat. Cell Mol. Genet. 7, 411-42 l, 198 I. PHILLIPS, P.D., PIGNOLO, R.J., and CRISTOFALO, V.J. Insulin-like growth factor-l: Specific binding to high and low at~nity sites and mitogenic action throughout the life span of W138 cells. J. Cell. Physiol. 133, 135-143, 1987. RABINOVITCH, P.S. and NORWOOD, T.H. Comparative heterokaryon study of cellular senescence and the serum-deprived state. Exp. Cell Res. 130, l0 l - 109, 1980. SESTER, U., SAWADA, M., and WANG, E. Purification and biochemical characterization ofstatin, a nonproliferation-specific protein from rat liver. J. Biol. Chem. 265, 19966-19972, 1990. SMITH, J.R. and HAYFLICK, L. Variation in the life-span of clones derived from human diploid cell strains. J. CellBiol. 62, 48-53, 1974. SMITH, J.R. and WHITNEY, R.G. Intraclonal variation in proliferative potential of human diploid fibroblasts: Stochastic mechanism for cellular aging. Science 207, 82-84, 1980. STEIN, G.H. and YANISHEVSKY, R.M. Quiescent human diploid cells can inhibit entry into S phase in replicative nuclei in heterodikaryons. Proc. Natl. Acad. Sci. U. S. A. 78, 3025-3029, 1981. TSANACLIS, A.M.C., BREM, S.S., GATELY, S., SCHIPPER, H.M., and WANG, E. Statin immunolocalization in human brain tumors, detection of noncyclingcells using a novel marker of cell quiescence. Cancer 68, 786-792, 1991. WANG, E. Rapid disappearance ofstatin, a nonproliferatingand senescent cell-specific protein, upon re-entering the process of cell cycling. J. CellBiol. 101, 1695-1701, 1985a. WANG, E. Contact-inhibition-inducedquiescent state is marked by intense nuclear expression ofstatin. J. Cell. Physiol. 133, 151-157, 1987. WANG, E. Statin, a nonproliferation-specific protein, is associated with the nuclear envelope and is heterogeneously distributed in cells leaving quiescent state. J. Cell. Physiol. 140, 418-426, 1989. WANG, E. and KRUEGER, J.G. Application of an unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissues. J. Histochem. Cytochem. 33, 587-594, 1985. WANG, E. and LIN, S.L. Disappearance of statin, a protein marker for nonproliferating and senescent cells, lollowing serum-stimulated cell cycle entry. Exp. Cell Res. 167, 135-143, 1986. WANG, E. and TOMASZEWSKI, G. The granular presence of terminin is the marker to distinguish between senescent and quiescent states. J. Cell Physiol. 147, 514-522, 1991. WANG, E.A. 57,000-mol-wt. protein uniquely present in nonproliferatingceils and senescent human fibroblasts. J. CellBiol. 100, 545-551, 1985b.