- Email: [email protected]
p202 Prevents Apoptosis in Murine AKR-2B Fibroblasts
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 379–382 (1998)
RC988804
p202 Prevents Apoptosis in Murine AKR-2B Fibroblasts Dimpy Koul, Ruth Lapushin, Hong-Ji Xu, Gordon B. Mills, Jordan U. Gutterman, and Divaker Choubey1 Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030
Received May 11, 1998
p202 is an interferon (IFN)-inducible, primarily nuclear, phosphoprotein (52-kDa) whose overexpression in transfected cells inhibits colony formation. p202 binds to the retinoblastoma tumor suppressor protein and two other members of the pocket family proteins (p107 and p130). Moreover, overexpression of p202 in transfected cells inhibits the transcriptional activity of E2Fs (E2F-1/DP-1 and E2F-4/DP-1), p53, AP-1 c-Fos and c-Jun, NF-kB p50 and p65. Here we demonstrate that inhibition of endogenous p202 production in murine AKR-2B fibroblasts did not result in an increase in cell proliferation. Instead, these cells exhibited increased susceptibility to apoptosis in response to decrease in serum concentrations in the growth medium. These observations are consistent with the notion that normal levels of p202 may be needed for the regulation of cell proliferation. q 1998 Academic Press
Interferons (IFNs) are multifunctional cytokines shown to affect apoptosis (1, 2). However, the role of IFN-inducible proteins in this process remains to be established. Earlier, we described an IFN-inducible, primarily nuclear phosphoprotein, p202 (52-kDa), whose levels are increased in cultured cells in response to treatment with IFN (3-5) or during differentiation of murine skeletal muscle cells in vitro (6). Moreover, constitutive overexpression of p202 in transfected cell lines inhibits colony formation (7-9; Choubey, unpublished data). In transfected cells p202 modulates activity of several transcription factors including E2F (E2F1/DP-1 and E2F-4/DP-1) (8, 10), p53 (11), NF-kB p50 and p65, AP-1 c-Fos and c-Jun (9), MyoD and myogenin (6). p202 binds to pRb and two other pocket proteins, p107 and p130 (7, 10). Binding of p202 to E2Fs (E2F1/DP-1 and E2F-4/DP-1) in complex with the pocket 1 To whom correspondence should be addressed at present address: Department of Radiotherapy, Loyola University Chicago-Hines VAMC 5th Avenue & Roosevelt Road (114-B), Hines, IL 60141. Fax: (708) 216-2647. E-mail: [email protected].
proteins correlates with the inhibition of the sequencespecific DNA-binding of E2F complexes (8, 10). To further investigate the functional role of p202 in cell growth regulation, we established stable cell lines of murine AKR-2B fibroblasts allowing tetracyclineregulated (12) expression of antisense RNA to the 202 gene. Using these cell models, we demonstrate that removal of tetracycline from the growth medium, which resulted in decreased endogenous levels of p202, in asynchronously growing cells inhibited cell proliferation and increased their susceptibility to apoptosis in response to a decrease in serum concentration in the growth medium. Taken together, these observations are consistent with the possibility that normal levels of p202 are needed for the regulation of cell proliferation. MATERIALS AND METHODS Cell line. Murine AKR 2-B embryonic fibroblasts (originally from Dr. Harold Moses, The Vanderbilt Cancer Center) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (13). Plasmid and transfections. The plasmid used for expression of an antisense RNA to murine 202 gene was constructed by ligating a 1.4 kb Bam H1 cDNA fragment from plasmid pCMV-202 (8) into the Bam H1 site of the EC1214A plasmid vector. EC 1214A is a single plasmid vector containing a modified tetracycline-regulatable gene expression system (12, 14, Xu et al., unpublished data). The resultant recombinant plasmid vector was named EC1214A-202AS. Uncloned AKR 2-B cells were transfected with EC1214A-202AS expression plasmid by calcium phosphate precipitation method (15). Cells were maintained in 4 mg/ml of tetracycline and colonies were selected using 500 mg/ml of G418. After about three weeks, isolated colonies were cloned by cylinder cloning. The clonal cell lines were tested by western blotting for inducible reduction in endogenous p202 protein levels. Two clonal cell lines (IAS-5 and IAS-12) were found to express low levels of p202 in presence of tetracycline (presumably due to some ‘‘leakiness’’ in the promoter) and in response to removal of Tet from the growth medium p202 levels decreased further. These two cell lines were maintained in DME medium supplemented with 100 mg/ml G418 and 4 mg/ml tetracycline and used for further studies. For experimental studies described in Figs. 2, 3, 4 and 5, IAS-12 cells were grown without tetracycline in the growth medium. Immunoblotting. Extracts from the parental AKR-2B cells (control or treated with IFN) or clonal cell lines (grown in the presence 0006-291X/98 $25.00
379
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8804
/
6955$$$941
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
conditions (e.g., confluence and serum concentration) of these cells. This is consistent with our earlier observations in which we found that endogenous levels of p202 are lower in quiescent BALB/c 3T3 cells and confluent AKR-2B cells (4). FIG. 1. Removal of tetracycline from the growth medium decreases endogenous levels of p202. IAS-12 cells (stably expressing antisense RNA to the 202 gene) were grown in DMEM (supplemented with 10% fetal bovine serum) without (lanes 4 and 6) or with (lanes 3 and 5) tetracycline for 48 hours. As a control for p202 size, cells of the parental AKR-2B cell line were also grown in DMEM (supplemented with 10% fetal bovine serum) and either left untreated (lane 1) or treated with IFN (lane 2) for 48 h as described in materials and methods. Total cell extracts were prepared as described in the materials and methods and analyzed by SDS-PAGE followed by immunoblotting using polyclonal anti-p202 antiserum. The bands were scanned, and the extent of p202 reduction in lanes 4 and 6 is indicated below the bands. The p202 protein band is indicated.
or absence of tetracycline) were prepared as described before (4). The extracts were analyzed by SDS-PAGE followed by immunoblotting using anti-p202 antiserum (4). Growth curves. For determination of cell proliferation rate, cells (AKR-2B or IAS-12) were seeded in DMEM supplemented with either 10% or 1% fetal bovine serum (Clontech, tested for the absence of tetracycline) at 21105 cells per well in 24 well plate (in triplicates). After two and half days, cells were washed with PBS and reefed with the same medium. Cells were counted twice on the indicated days. Flow cytometry analysis. For cell cycle analysis, 2.51105 cells were seeded per 100-mm plate and were grown in DMEM containing either 10% or 1% fetal bovine serum. At indicated times, cells were trypsinized, washed with PBS and fixed with 2 ml of 70% ethanol overnight. For FACS analysis, the fixed cells were centrifuged at 1,500 1 g for 5 minutes and resuspended in 1 ml of PBS solution containing 50 mg/ml of RNase (from Sigma) and propidium iodide (from Sigma). The stained cells were analyzed by flow cytometry (Coulter Epics Profile, Miami, FL). The percentage of cells in various cell cycle phases was determined by using combination of multicycle and Epics-Elite software program (Phoenix Flow Systems, San Diego, CA).
Reduction in p202 inhibits cell proliferation. Since overexpression of p202 in transfected cells was found to retard cell proliferation (7-9), to examine the effects of reduced levels of p202 on cell proliferation, we compared the growth characteristics of IAS-12 cells with that of parental AKR-2B cells. For this purpose, equal number of AKR-2B or IAS-12 cells were seeded (in triplicates) in growth medium supplemented with either 10% fetal bovine serum or 1% fetal bovine serum (under these conditions endogenous levels of p202 are lower in AKR-2B cells; ref. 4) and counted cells on the indicated days. As seen in Fig. 2, parental AKR-2B cells continued to proliferate normally in growth medium supplemented with 10% serum whereas, surprisingly, IAS-12 cells did not proliferate significantly during this period. Interestingly, the parental AKR-2B cells increased in number from day 1 to day 3 in medium supplemented with 1% serum whereas IAS-12 cells decreased significantly in number after two days of incubation. Furthermore, cultures of IAS-12 cells grown in the presence of 1% serum contained a significant number of floating cells which appeared to contain condensed chromatin (Fig. 3, compare panel B with D). Similar results were obtained with the stable cell line, IAS-5 (data not shown). To determine whether the decrease in cell number
DNA-fragmentation assay. DNA-fragmentation assay was done as described previously (16).
RESULTS AND DISCUSSION Regulated reduction in endogenous levels of p202. To study the functional role of p202 in cell proliferation, we established stable cell lines of murine AKR-2B fibroblasts allowing tetracycline-regulated (12) decrease in endogenous levels of p202. Several G418-resistant stable clonal cell lines were screened by immunoblotting for reduction in endogenous levels of p202 in response to the removal of Tet from the growth medium. On this basis, we selected two stable cell lines (IAS-5 and IAS-12) which reproducibly showed an inducible reduction (about five-fold) in endogenous levels of p202 in extracts prepared from asynchronously growing subconfluent cultures of cells (Fig. 1, compare lane 3 with 4 or lane 5 with 6). Furthermore, the extent of decrease in endogenous levels of p202 was dependent on culture
FIG. 2. Decrease in endogenous levels of p202 retard cell proliferation. IAS-12 cells were grown (21105 cells per well, in triplicates, in 24 well plate) in DMEM supplemented with either 10% (c) or 1% (d) fetal bovine serum without tetracycline. As a control, equal number of the parental AKR-2B cells were also grown either in 10% (a) or 1% (b) serum. Cells were trypsinized on the indicated days and counted (twice) using cell counting chamber. The data shown are an arithmetic mean of three independent experiments.
380
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Decrease in endogenous levels of p202 promotes cell death. IAS-12 cells (panel C and D) were grown in DMEM supplemented with 10% (panel C) or 1% serum (panel D) without Tet. As a control, cells of the parental AKR-2B cell line (panel A and B) were also grown in DMEM supplemented with 10% (panel A) or 1% serum (panel B). After three days cells were photographed for alterations in their morphology.
seen in cultures of IAS-12 cells (grown in presence of 1% serum) in the above experiments was due to cell death, we analyzed these cells on the day 3 by flow cytometry. As shown in Fig. 4, AKR-2B cells grown in medium supplemented with either 10% (panel A) or 1% (panel C) fetal bovine serum did not appear to contain significant number of cells in sub-G0 (hypodiploid, an indication of apoptosis). Intriguingly, IAS-12 cells under 1% serum growth conditions had a marked increase (40-50%) in the number of hypodiploid cells. DNA-fragmentation analysis (Fig. 5) revealed that incubation of IAS-12 cells, but not AKR-2B cells, in 1% serum correlated with appearance of a typical oligonucleosomal DNA-ladder (Figure 5, compare lane 1 with 2). These observations suggest that reduced expression of p202, under the growth conditions tested, leads to an increased rate of programmed cell death by apoptosis. IFNs have been shown to induce cell death by direct cytotoxic effect in primary tumor cells from some malignancies (17, 18), and in some instances the cytoreductive effect of IFN has been shown to be due to the induction of apoptosis (1, 2, 19-21). In some systems
FIG. 4. IAS-12 cells undergo cell death in reduced serum concentrations. IAS-12 cells were grown in DMEM supplemented with 10% (panel B) or 1% serum (panel D) without Tet. As a control, cells of the parental AKR-2B cell line were also grown in DMEM supplemented with 10% (panel A) or 1% serum (panel C). After three days, cell were fixed and analyzed by flow cytometry using combination of multicycle and Epics-Elite software program.
381
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
FIG. 5. Decreased serum concentrations induce DNA-fragmentation in IAS-12 cells. IAS-12 cells were grown in DMEM supplemented with 1% serum without Tet (lane 2). As a control, cells of the parental AKR-2B cells were also grown in DMEM supplemented with 1% serum (lane 1). After three days when IAS-12 cultures contained morphologically apoptotic cells, cultures were processed for DNAfragmentation assay as described in methods. M: 100 bp doublestranded DNA-ladder.
IFN can protect malignant cells from spontaneous apoptosis as well as apoptosis induced by cytotoxic agents or by wild-type p53 (22, 23). Moreover, little is known about the reason for the variable sensitivity to IFN-induced apoptosis between different cell lineages. IFNs do not appear to alter the expression of Bcl-2 or Bax protein in cells sensitive to IFN-induced apoptosis and apoptosis seems to occur independently of a wildtype p53 protein (21). Overexpression of p202 in transfected cells retards colony formation (7-9). Results presented in this study underscore p202’s role in the regulation of cell proliferation and raise the possibility that appropriate levels of p202 may be needed for normal regulation of cell proliferation. Although, the molecular basis of inhibition of cell proliferation by reduced endogenous levels of p202 remains to be explored, it is conceivable that lower cellular levels of p202 (p202 inhibits the transcriptional activity of E2F and AP1; ref. 8 and 9) may result in increased activity of E2F and c-Jun. This in turn may contribute to increased sensitivity to apoptosis under low serum conditions (24-27). Since pRb, another negative regulator of E2F’s activity, has been shown to have an antiapoptotic activity in some cells (28, 29), it is conceivable that lower levels of p202 may promote apoptosis through a similar mechanism. ACKNOWLEDGMENTS Research conducted was supported by a grant (CA69031) from the National Institutes of Health (to D.C.) and, in part, by funds from the Clayton Foundation for Research (to J.U.G.).
1. Grander, D., Sangfelt, O., and Erickson, S. (1997) Eur. J. Haematol. 59, 129–135. 2. Jewell, A. P. (1996) Leukemia Lymphoma 21, 43 ff. 3. Choubey, D., Snoddy, J., Chaturvedi, V., Toniato, E., Opdenakker, G., Thakur, A., Samanta, H., Engel, D., and Lengyel, P. (1989) J. Biol. Chem. 264, 17182–17189. 4. Choubey, D., and Lengyel, P. (1993) J. Interferon Res. 13, 43–52. 5. Lengyel, P., Choubey, D., Li, S.-J., and Datta, B. (1996) Sem. Virol. 6, 203–213. 6. Datta, B., Min, W., Burma, S., and Lengyel, P. (1998) Mol. Cell. Biol. 18, 1074–1083. 7. Choubey, D., and Lengyel, P. (1995) J. Biol. Chem. 270, 6134– 6140. 8. Choubey, D., Li, S. J., Datta, B., Gutterman, J. U., and Lengyel, P. (1996) EMBO J. 15, 5668–5678. 9. Min, W., Ghosh, S., and Lengyel, P. (1996) Mol. Cell. Biol. 16, 359–368. 10. Choubey, D., and Gutterman, J. U. (1997) Oncogene 15, 291–302. 11. Datta, B., Li, B., Choubey, D., Nallur, G., and Lengyel, P. (1997) J. Biol. Chem. 271, 27544–27557. 12. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551. 13. Choubey, D., and Lengyel, P. (1992) J. Cell Biol. 116, 1333– 1341. 14. Xu, H.-J., Zhou, Y. L., Ji, W., Perng, G. S., Kruzelock, R., Bast, R. C., Mills, G. B., Li, J., and Hu, S. X. (1997) Oncogene 15, 2589–2596. 15. Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Lab. Press, New York, USA. 16. Kondo, S., Barnett, G. H., Hara, H., Morimura, A. T., and Takeuchi, J. (1995) Oncogene 10, 2001–2006. 17. Grander, D., Xu, B., and Einhorn, S. (1993) Eur. J. Cancer 14, 1940–1943. 18. Manabe, A., Yi, T., Kumagi, M., and Campara, D. (1993) Leukemia 7, 1990–1995. 19. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., and Kimchi, A. (1995) Genes Dev. 9, 15–30. 20. Rodriguez-Villanueva, J., and McDonnell, T. J. (1995) Int. J. Cancer 61, 110–114. 21. Sangfelt, O., Erickson, S., Einhorn, S., Castro, J., Heiden, T., and Grander, D. (1997) Cell Growth Diff. 8, 343–352. 22. Sangfelt, O., Einhorn, S., Bjorklund, A. C., Wiman, K. G., Okan, I., and Grander, D. (1996) Int. J. Cancer 67, 106–112. 23. Milner, A. E., Grand, R. J., and Gregory, C. D. (1995) Int. J. Cancer 61, 348–354. 24. Quin, X. Q., Livingston, D. M., Kaelin, W. G., and Adams, P. D. (1994) Proc. Natl. Acad. Sci. USA 91, 10918–10922. 25. Wu, X., and Levine, A. J. (1994) Proc. Natl. Acad. Sci. USA 91, 3602–3606. 26. Bargou, R. C., Wagner, C., Bommert, K., Arnold, W., Daniel, P. T., Mapara, M. Y., Grinstein, E., Royer, H. D., and Darken, B. (1996) J. Exp. Med. 183, 1205–1213. 27. Bossy-wetzel, E., Bakiri, L., and Yaniv, M. (1997) EMBO J. 16, 1695–1709. 28. Kastan, M. M., and Giordano, A. (1998) Cell Death Differ. 5, 132–140. 29. Berry, D. E., Lu, Y., Schmidt, B., Fallon, P. G., O’Connell, C., Hu, S.-X., Xu, H.-J., and Blanck, G. (1996) Oncogene 12, 1809– 1819.
382
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC
247, 379–382 (1998)
RC988804
p202 Prevents Apoptosis in Murine AKR-2B Fibroblasts Dimpy Koul, Ruth Lapushin, Hong-Ji Xu, Gordon B. Mills, Jordan U. Gutterman, and Divaker Choubey1 Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030
Received May 11, 1998
p202 is an interferon (IFN)-inducible, primarily nuclear, phosphoprotein (52-kDa) whose overexpression in transfected cells inhibits colony formation. p202 binds to the retinoblastoma tumor suppressor protein and two other members of the pocket family proteins (p107 and p130). Moreover, overexpression of p202 in transfected cells inhibits the transcriptional activity of E2Fs (E2F-1/DP-1 and E2F-4/DP-1), p53, AP-1 c-Fos and c-Jun, NF-kB p50 and p65. Here we demonstrate that inhibition of endogenous p202 production in murine AKR-2B fibroblasts did not result in an increase in cell proliferation. Instead, these cells exhibited increased susceptibility to apoptosis in response to decrease in serum concentrations in the growth medium. These observations are consistent with the notion that normal levels of p202 may be needed for the regulation of cell proliferation. q 1998 Academic Press
Interferons (IFNs) are multifunctional cytokines shown to affect apoptosis (1, 2). However, the role of IFN-inducible proteins in this process remains to be established. Earlier, we described an IFN-inducible, primarily nuclear phosphoprotein, p202 (52-kDa), whose levels are increased in cultured cells in response to treatment with IFN (3-5) or during differentiation of murine skeletal muscle cells in vitro (6). Moreover, constitutive overexpression of p202 in transfected cell lines inhibits colony formation (7-9; Choubey, unpublished data). In transfected cells p202 modulates activity of several transcription factors including E2F (E2F1/DP-1 and E2F-4/DP-1) (8, 10), p53 (11), NF-kB p50 and p65, AP-1 c-Fos and c-Jun (9), MyoD and myogenin (6). p202 binds to pRb and two other pocket proteins, p107 and p130 (7, 10). Binding of p202 to E2Fs (E2F1/DP-1 and E2F-4/DP-1) in complex with the pocket 1 To whom correspondence should be addressed at present address: Department of Radiotherapy, Loyola University Chicago-Hines VAMC 5th Avenue & Roosevelt Road (114-B), Hines, IL 60141. Fax: (708) 216-2647. E-mail: [email protected].
proteins correlates with the inhibition of the sequencespecific DNA-binding of E2F complexes (8, 10). To further investigate the functional role of p202 in cell growth regulation, we established stable cell lines of murine AKR-2B fibroblasts allowing tetracyclineregulated (12) expression of antisense RNA to the 202 gene. Using these cell models, we demonstrate that removal of tetracycline from the growth medium, which resulted in decreased endogenous levels of p202, in asynchronously growing cells inhibited cell proliferation and increased their susceptibility to apoptosis in response to a decrease in serum concentration in the growth medium. Taken together, these observations are consistent with the possibility that normal levels of p202 are needed for the regulation of cell proliferation. MATERIALS AND METHODS Cell line. Murine AKR 2-B embryonic fibroblasts (originally from Dr. Harold Moses, The Vanderbilt Cancer Center) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (13). Plasmid and transfections. The plasmid used for expression of an antisense RNA to murine 202 gene was constructed by ligating a 1.4 kb Bam H1 cDNA fragment from plasmid pCMV-202 (8) into the Bam H1 site of the EC1214A plasmid vector. EC 1214A is a single plasmid vector containing a modified tetracycline-regulatable gene expression system (12, 14, Xu et al., unpublished data). The resultant recombinant plasmid vector was named EC1214A-202AS. Uncloned AKR 2-B cells were transfected with EC1214A-202AS expression plasmid by calcium phosphate precipitation method (15). Cells were maintained in 4 mg/ml of tetracycline and colonies were selected using 500 mg/ml of G418. After about three weeks, isolated colonies were cloned by cylinder cloning. The clonal cell lines were tested by western blotting for inducible reduction in endogenous p202 protein levels. Two clonal cell lines (IAS-5 and IAS-12) were found to express low levels of p202 in presence of tetracycline (presumably due to some ‘‘leakiness’’ in the promoter) and in response to removal of Tet from the growth medium p202 levels decreased further. These two cell lines were maintained in DME medium supplemented with 100 mg/ml G418 and 4 mg/ml tetracycline and used for further studies. For experimental studies described in Figs. 2, 3, 4 and 5, IAS-12 cells were grown without tetracycline in the growth medium. Immunoblotting. Extracts from the parental AKR-2B cells (control or treated with IFN) or clonal cell lines (grown in the presence 0006-291X/98 $25.00
379
Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 8804
/
6955$$$941
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
conditions (e.g., confluence and serum concentration) of these cells. This is consistent with our earlier observations in which we found that endogenous levels of p202 are lower in quiescent BALB/c 3T3 cells and confluent AKR-2B cells (4). FIG. 1. Removal of tetracycline from the growth medium decreases endogenous levels of p202. IAS-12 cells (stably expressing antisense RNA to the 202 gene) were grown in DMEM (supplemented with 10% fetal bovine serum) without (lanes 4 and 6) or with (lanes 3 and 5) tetracycline for 48 hours. As a control for p202 size, cells of the parental AKR-2B cell line were also grown in DMEM (supplemented with 10% fetal bovine serum) and either left untreated (lane 1) or treated with IFN (lane 2) for 48 h as described in materials and methods. Total cell extracts were prepared as described in the materials and methods and analyzed by SDS-PAGE followed by immunoblotting using polyclonal anti-p202 antiserum. The bands were scanned, and the extent of p202 reduction in lanes 4 and 6 is indicated below the bands. The p202 protein band is indicated.
or absence of tetracycline) were prepared as described before (4). The extracts were analyzed by SDS-PAGE followed by immunoblotting using anti-p202 antiserum (4). Growth curves. For determination of cell proliferation rate, cells (AKR-2B or IAS-12) were seeded in DMEM supplemented with either 10% or 1% fetal bovine serum (Clontech, tested for the absence of tetracycline) at 21105 cells per well in 24 well plate (in triplicates). After two and half days, cells were washed with PBS and reefed with the same medium. Cells were counted twice on the indicated days. Flow cytometry analysis. For cell cycle analysis, 2.51105 cells were seeded per 100-mm plate and were grown in DMEM containing either 10% or 1% fetal bovine serum. At indicated times, cells were trypsinized, washed with PBS and fixed with 2 ml of 70% ethanol overnight. For FACS analysis, the fixed cells were centrifuged at 1,500 1 g for 5 minutes and resuspended in 1 ml of PBS solution containing 50 mg/ml of RNase (from Sigma) and propidium iodide (from Sigma). The stained cells were analyzed by flow cytometry (Coulter Epics Profile, Miami, FL). The percentage of cells in various cell cycle phases was determined by using combination of multicycle and Epics-Elite software program (Phoenix Flow Systems, San Diego, CA).
Reduction in p202 inhibits cell proliferation. Since overexpression of p202 in transfected cells was found to retard cell proliferation (7-9), to examine the effects of reduced levels of p202 on cell proliferation, we compared the growth characteristics of IAS-12 cells with that of parental AKR-2B cells. For this purpose, equal number of AKR-2B or IAS-12 cells were seeded (in triplicates) in growth medium supplemented with either 10% fetal bovine serum or 1% fetal bovine serum (under these conditions endogenous levels of p202 are lower in AKR-2B cells; ref. 4) and counted cells on the indicated days. As seen in Fig. 2, parental AKR-2B cells continued to proliferate normally in growth medium supplemented with 10% serum whereas, surprisingly, IAS-12 cells did not proliferate significantly during this period. Interestingly, the parental AKR-2B cells increased in number from day 1 to day 3 in medium supplemented with 1% serum whereas IAS-12 cells decreased significantly in number after two days of incubation. Furthermore, cultures of IAS-12 cells grown in the presence of 1% serum contained a significant number of floating cells which appeared to contain condensed chromatin (Fig. 3, compare panel B with D). Similar results were obtained with the stable cell line, IAS-5 (data not shown). To determine whether the decrease in cell number
DNA-fragmentation assay. DNA-fragmentation assay was done as described previously (16).
RESULTS AND DISCUSSION Regulated reduction in endogenous levels of p202. To study the functional role of p202 in cell proliferation, we established stable cell lines of murine AKR-2B fibroblasts allowing tetracycline-regulated (12) decrease in endogenous levels of p202. Several G418-resistant stable clonal cell lines were screened by immunoblotting for reduction in endogenous levels of p202 in response to the removal of Tet from the growth medium. On this basis, we selected two stable cell lines (IAS-5 and IAS-12) which reproducibly showed an inducible reduction (about five-fold) in endogenous levels of p202 in extracts prepared from asynchronously growing subconfluent cultures of cells (Fig. 1, compare lane 3 with 4 or lane 5 with 6). Furthermore, the extent of decrease in endogenous levels of p202 was dependent on culture
FIG. 2. Decrease in endogenous levels of p202 retard cell proliferation. IAS-12 cells were grown (21105 cells per well, in triplicates, in 24 well plate) in DMEM supplemented with either 10% (c) or 1% (d) fetal bovine serum without tetracycline. As a control, equal number of the parental AKR-2B cells were also grown either in 10% (a) or 1% (b) serum. Cells were trypsinized on the indicated days and counted (twice) using cell counting chamber. The data shown are an arithmetic mean of three independent experiments.
380
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Decrease in endogenous levels of p202 promotes cell death. IAS-12 cells (panel C and D) were grown in DMEM supplemented with 10% (panel C) or 1% serum (panel D) without Tet. As a control, cells of the parental AKR-2B cell line (panel A and B) were also grown in DMEM supplemented with 10% (panel A) or 1% serum (panel B). After three days cells were photographed for alterations in their morphology.
seen in cultures of IAS-12 cells (grown in presence of 1% serum) in the above experiments was due to cell death, we analyzed these cells on the day 3 by flow cytometry. As shown in Fig. 4, AKR-2B cells grown in medium supplemented with either 10% (panel A) or 1% (panel C) fetal bovine serum did not appear to contain significant number of cells in sub-G0 (hypodiploid, an indication of apoptosis). Intriguingly, IAS-12 cells under 1% serum growth conditions had a marked increase (40-50%) in the number of hypodiploid cells. DNA-fragmentation analysis (Fig. 5) revealed that incubation of IAS-12 cells, but not AKR-2B cells, in 1% serum correlated with appearance of a typical oligonucleosomal DNA-ladder (Figure 5, compare lane 1 with 2). These observations suggest that reduced expression of p202, under the growth conditions tested, leads to an increased rate of programmed cell death by apoptosis. IFNs have been shown to induce cell death by direct cytotoxic effect in primary tumor cells from some malignancies (17, 18), and in some instances the cytoreductive effect of IFN has been shown to be due to the induction of apoptosis (1, 2, 19-21). In some systems
FIG. 4. IAS-12 cells undergo cell death in reduced serum concentrations. IAS-12 cells were grown in DMEM supplemented with 10% (panel B) or 1% serum (panel D) without Tet. As a control, cells of the parental AKR-2B cell line were also grown in DMEM supplemented with 10% (panel A) or 1% serum (panel C). After three days, cell were fixed and analyzed by flow cytometry using combination of multicycle and Epics-Elite software program.
381
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC
Vol. 247, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
FIG. 5. Decreased serum concentrations induce DNA-fragmentation in IAS-12 cells. IAS-12 cells were grown in DMEM supplemented with 1% serum without Tet (lane 2). As a control, cells of the parental AKR-2B cells were also grown in DMEM supplemented with 1% serum (lane 1). After three days when IAS-12 cultures contained morphologically apoptotic cells, cultures were processed for DNAfragmentation assay as described in methods. M: 100 bp doublestranded DNA-ladder.
IFN can protect malignant cells from spontaneous apoptosis as well as apoptosis induced by cytotoxic agents or by wild-type p53 (22, 23). Moreover, little is known about the reason for the variable sensitivity to IFN-induced apoptosis between different cell lineages. IFNs do not appear to alter the expression of Bcl-2 or Bax protein in cells sensitive to IFN-induced apoptosis and apoptosis seems to occur independently of a wildtype p53 protein (21). Overexpression of p202 in transfected cells retards colony formation (7-9). Results presented in this study underscore p202’s role in the regulation of cell proliferation and raise the possibility that appropriate levels of p202 may be needed for normal regulation of cell proliferation. Although, the molecular basis of inhibition of cell proliferation by reduced endogenous levels of p202 remains to be explored, it is conceivable that lower cellular levels of p202 (p202 inhibits the transcriptional activity of E2F and AP1; ref. 8 and 9) may result in increased activity of E2F and c-Jun. This in turn may contribute to increased sensitivity to apoptosis under low serum conditions (24-27). Since pRb, another negative regulator of E2F’s activity, has been shown to have an antiapoptotic activity in some cells (28, 29), it is conceivable that lower levels of p202 may promote apoptosis through a similar mechanism. ACKNOWLEDGMENTS Research conducted was supported by a grant (CA69031) from the National Institutes of Health (to D.C.) and, in part, by funds from the Clayton Foundation for Research (to J.U.G.).
1. Grander, D., Sangfelt, O., and Erickson, S. (1997) Eur. J. Haematol. 59, 129–135. 2. Jewell, A. P. (1996) Leukemia Lymphoma 21, 43 ff. 3. Choubey, D., Snoddy, J., Chaturvedi, V., Toniato, E., Opdenakker, G., Thakur, A., Samanta, H., Engel, D., and Lengyel, P. (1989) J. Biol. Chem. 264, 17182–17189. 4. Choubey, D., and Lengyel, P. (1993) J. Interferon Res. 13, 43–52. 5. Lengyel, P., Choubey, D., Li, S.-J., and Datta, B. (1996) Sem. Virol. 6, 203–213. 6. Datta, B., Min, W., Burma, S., and Lengyel, P. (1998) Mol. Cell. Biol. 18, 1074–1083. 7. Choubey, D., and Lengyel, P. (1995) J. Biol. Chem. 270, 6134– 6140. 8. Choubey, D., Li, S. J., Datta, B., Gutterman, J. U., and Lengyel, P. (1996) EMBO J. 15, 5668–5678. 9. Min, W., Ghosh, S., and Lengyel, P. (1996) Mol. Cell. Biol. 16, 359–368. 10. Choubey, D., and Gutterman, J. U. (1997) Oncogene 15, 291–302. 11. Datta, B., Li, B., Choubey, D., Nallur, G., and Lengyel, P. (1997) J. Biol. Chem. 271, 27544–27557. 12. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551. 13. Choubey, D., and Lengyel, P. (1992) J. Cell Biol. 116, 1333– 1341. 14. Xu, H.-J., Zhou, Y. L., Ji, W., Perng, G. S., Kruzelock, R., Bast, R. C., Mills, G. B., Li, J., and Hu, S. X. (1997) Oncogene 15, 2589–2596. 15. Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Lab. Press, New York, USA. 16. Kondo, S., Barnett, G. H., Hara, H., Morimura, A. T., and Takeuchi, J. (1995) Oncogene 10, 2001–2006. 17. Grander, D., Xu, B., and Einhorn, S. (1993) Eur. J. Cancer 14, 1940–1943. 18. Manabe, A., Yi, T., Kumagi, M., and Campara, D. (1993) Leukemia 7, 1990–1995. 19. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., and Kimchi, A. (1995) Genes Dev. 9, 15–30. 20. Rodriguez-Villanueva, J., and McDonnell, T. J. (1995) Int. J. Cancer 61, 110–114. 21. Sangfelt, O., Erickson, S., Einhorn, S., Castro, J., Heiden, T., and Grander, D. (1997) Cell Growth Diff. 8, 343–352. 22. Sangfelt, O., Einhorn, S., Bjorklund, A. C., Wiman, K. G., Okan, I., and Grander, D. (1996) Int. J. Cancer 67, 106–112. 23. Milner, A. E., Grand, R. J., and Gregory, C. D. (1995) Int. J. Cancer 61, 348–354. 24. Quin, X. Q., Livingston, D. M., Kaelin, W. G., and Adams, P. D. (1994) Proc. Natl. Acad. Sci. USA 91, 10918–10922. 25. Wu, X., and Levine, A. J. (1994) Proc. Natl. Acad. Sci. USA 91, 3602–3606. 26. Bargou, R. C., Wagner, C., Bommert, K., Arnold, W., Daniel, P. T., Mapara, M. Y., Grinstein, E., Royer, H. D., and Darken, B. (1996) J. Exp. Med. 183, 1205–1213. 27. Bossy-wetzel, E., Bakiri, L., and Yaniv, M. (1997) EMBO J. 16, 1695–1709. 28. Kastan, M. M., and Giordano, A. (1998) Cell Death Differ. 5, 132–140. 29. Berry, D. E., Lu, Y., Schmidt, B., Fallon, P. G., O’Connell, C., Hu, S.-X., Xu, H.-J., and Blanck, G. (1996) Oncogene 12, 1809– 1819.
382
AID
BBRC 8804
/
6955$$$942
06-04-98 09:12:53
bbrcg
AP: BBRC