- Email: [email protected]
Effects of HRAS Oncogene on Cell Cycle Progression in a Cervical Cancer-Derived Cell Line
Archives of Medical Research 36 (2005) 311–316
ORIGINAL ARTICLE
Effects of HRAS Oncogene on Cell Cycle Progression in a Cervical Cancer-Derived Cell Line Emilio Co´rdova-Alarco´n,a Federico Centeno,b Jorge Reyes-Esparza,c Alejandro Garcı´a-Carranca´d,e and Efraı´n Garridob a
Departamento de Biomedicina Molecular, bDepartamento de Gene´tica y Biologı´a Molecular, Centro de Investigacio´n y de Estudios Avanzados del Instituto Polite´cnico Nacional, Me´xico, D.F., Me´xico c Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Me´xico d Unidad de Investigacio´n Biome´dica en Ca´ncer, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Me´xico, D.F., Me´xico e Instituto Nacional de Cancerologı´a, Secretarı´a de Salud, Me´xico, D.F., Me´xico Received for publication March 5, 2004; accepted July 12, 2004 (04/043).
Background. Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes. Methods. HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways. Results. We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase. Conclusions. Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells. 쑖 2005 IMSS. Published by Elsevier Inc. Key Words: Cervical carcinoma, HPV, RAS, Flow cytometry.
Introduction
Address reprint requests to: Dr. Efraı´n Garrido, Departamento de Gene´tica y Biologı´a Molecular, CINVESTAV-IPN, Av. IPN No. 2508, Col. San Pedro Zacatenco, 07360 Me´xico D.F., Me´xico. Phone: (⫹52) (55) 50613800 ext. 5373; FAX: (⫹52) (55) 5061-3800 ext. 5372; E-mail: [email protected]
0188-4409/05 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 5 .0 4 .0 01
Cervical carcinoma accounts for about 10% of all newly diagnosed cancers in women worldwide (1) and is one of the most frequent tumors in Mexico (2). Human papillomavirus infection (HPV) is the main factor associated with cervical carcinoma (3). The HPV gene products E6 and E7 play a critical role in cervical carcinogenesis by interfering with
쑖 2005 IMSS. Published by Elsevier Inc.
312
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
p53 and Rb functions, respectively, and deregulating the cell cycle (4). It has been realized that HPV infection is not sufficient for cervical carcinoma development, and that the presence of additional factors is needed (3). RAS proto-oncogenes are among the most frequently mutated genes in different human tumors (5,6). Mutations in RAS genes have been shown to coexist with oncogenic HPV in cervical cancer (7). In addition, RAS mutations are associated with a faster progression of HPV-induced lesions of uterine cervix (8). In mammals there are at least three RAS genes: NRAS, KRAS, and HRAS and the corresponding proteins function as regulators of different proliferation and survival signals (9). Although several putative RAS effectors have been reported, the RAF/MAPK-ERK kinase (MEK)/ extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/AKT pathways have been described as the most important (10,11). Although it has been described that in the absence of a functional Rb protein all the effects of RAS in cell cycle regulation are lost (12,13), recently several reports suggest that RAS have additional functions on cell cycle progression (14–16). In fact, neutralizing antibodies against Ras protein still inhibit DNA replication in Rb knockout cells (14,15). Additionally, it has been shown that MYC and RAS cooperate to induce cell cycle progression into S phase in an Rb-independent manner (16). These observations suggest that RAS oncogene could regulate G1/S progression independently of Rb protein. Therefore, despite the absence of a functional Rb protein in cervical cancer, RAS could participate in the development of this kind of tumor. In this work, we investigated whether the overexpression of oncogenic HRAS could alter cell cycle progression in the HeLa cell line containing the HPV-18 genome. We found that HRAS overexpression reduced G1 phase duration. Moreover, the G1/S transition was faster in overexpressing HRAS HeLa cells. Finally, using RAS-signaling chemical inhibitors, we showed that RAF/MEK/ERK and PI3K/AKT pathways participate in the G1 phase reduction mediated by HRAS.
Materials and Methods Cell culture. HeLa cells were stably transfected with plasmid pSV2neo HRAS Val12 (HeLa-EJ) or pSV2neo empty vector (HeLa-neo) and have already been described (17,18). Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM) and 10% newborn calf serum at 37⬚C in 5% CO2 atmosphere. Experiments were performed on subconfluent cultures not less than 24 h after plating. Drug treatments. The different treatments were performed at a cell density of 3.5 × 105 cells per 60-mm plate. Thymidine [50-89-5] (Amersham, Piscataway, NJ) was used at
2 mM (final concentration) for 24 h, and the cells were trypsinized at different times post-treatment. For mitotic arrest, cells were incubated for 24 h with 2 mM thymidine, washed and nocodazole [31430–18–9] (Sigma, St. Louis, MO) was added at 0.1 µg/mL for 15 h. Floating cells from the supernatant (mitotic cells) were recovered, washed out extensively, re-seeded and harvested at different time intervals. HeLa-EJ and neo cells were synchronized at mitosis as described above. After releasing them from mitotic arrest, different concentrations of the chemical inhibitors PD 098059 [167869–21–8] (Sigma) or wortmannin [19545–26– 7] (Sigma) were added and cells harvested and analyzed by flow cytometry. Flow cytometry analysis. One million cells treated or not with the different drugs were pelleted by low-speed centrifugation and the pellet resuspended with PBS. One mL of icecold 80% ethanol was added and the cell suspension was incubated overnight at 4⬚C. Fixed cells were harvested by centrifugation, resuspended in PBS containing RNase A (100 µg/mL) and propidium iodide (50 µg/mL), then incubated in the dark at 4⬚C for 1 h. The ethanol-fixed and propidium iodide-stained cells were analyzed for their DNA content in a FACS sort flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) (BDIS). Data analysis was performed using CellQuest (BDIS) and Modfit software (Verity Software House, Topsham, ME). Cell lysates and Western blot. Cells were harvested by trypsinization, washed with PBS and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4) supplemented with a mixture of phosphatase and protease inhibitors (1 mM sodium orthovanadate and 2 mM sodium fluoride, 1 mM PMSF, aprotinin 100 µg/mL, leupeptin 100 µg/mL, pepstatin 10 µg/mL, benzamidin 500 µg/mL, antipain 50 µg/mL and chymostatin 50 µg/mL). After incubation for 30 min on ice, lysates were cleared by centrifugation at 10,000 rpm for 10 min and protein concentration quantified with BioRad DC protein assay (BioRad, Hercules, CA). Equal amounts of proteins from the different samples were electrophoretically separated on a denaturating polyacrylamide gel (SDS-PAGE). Proteins were transferred to Hybond membrane (Amersham) and blocked with 5% non-fat milk in PBS. Membranes were incubated with anti-phosphospecific ERK antibodies (Cell Signaling, Beverly, MA), followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (Zymed, South San Francisco, CA). Proteins were observed using the ECL detection system (Amersham) followed by exposure to XOMAT films (Kodak, Rochester, NY). Membranes were stripped and re-blotted with anti-ERK antibodies (Cell Signaling). Statistical analysis. The statistical significance of the difference between intergroup comparisons was obtained using
HRAS Induces G1/S Progression in HeLa Cells
Student’s t-test. Data were expressed as mean ⫾ SD and were representative of at least three independent experiments.
Expression of exogenous HRAS results in the shortening of G1 phase duration in an HPV expressing cell line. In order to evaluate if HRAS oncogene can further affect cell cycle progression in the presence of HPV, we synchronized HeLa cells (a cell line derived from a human cervical carcinoma that expresses E6 and E7 from HPV-18), stably transfected with HRAS oncogene (HeLa-EJ) or empty vector (HeLa-neo). Initially, cell cultures were synchronized in mitosis (by thymidine–nocodazole treatment) and cell percentage in G1 phase was determined several hours after releasing the cells from the arrest. During the first 10 h after the release from the mitotic arrest, most of the HeLa-neo and HeLa-EJ cells were in G1 phase (⬎90%). However, after 12 h, the percentage of HeLa-EJ cells in G1 phase was consistently reduced in comparison with HeLa-neo cells. About 16 h later, while HeLa-neo cells were at the beginning of S phase, HeLa-EJ cells were already at the end of the same phase (Figure 1). Cell cycle reduction induced by HRAS occurred in late G1. An excess of thymidine is able to induce a reversible cell cycle arrest in G1/S in cell lines from different origins (including HeLa) (19). Incubation of cell cultures with 2 mM thymidine for 24 h induced a cell cycle arrest in G1/S in both HeLa-neo and HeLa-EJ cells (Figure 2). However, 2 h after thymidine was removed from the cultures, more than 80% of HeLa-EJ cells were in S phase. A similar percentage of HeLa-neo cells were in S phase only 6 h after removing the thymidine (Figure 2). BrdU incorporation assays were performed in order to determine DNA synthesis rate after thymidine arrest. These assays confirmed that DNA synthesis began in HeLa-EJ cells only 2 h after thymidine removal, whereas in HeLa-neo cells DNA synthesis began 4 h after thymidine was removed (Figure 3).
100
Percentage of cells in S
Results
313
**
**
75
50
25
0 0
2
4
6
8
10
12
hours Figure 2. HeLa cells stably transfected with HRAS show a faster G1/S progression. Cell cultures were incubated with 2 mM thymidine for 24 h and DNA content was determined several hours post-treatment by flow cytometry. Time 0 corresponds to 24 h of incubation with 2 mM thymidine. Data represent the mean value ⫾ SD. (**p ⬍0.01). HeLa-neo (䊐), HeLaEJ ( ).
The shortening of G1 phase induced by HRAS is dependent on RAF/MEK/ERK and PI3K/AKT pathways. The RAF/ MEK/ERK or PI3K/AKT pathways mediate the expression effects of HRAS on cell cycle regulation in different cell types (20). Therefore, we assessed the implication of these pathways in the HRAS-dependent reduction of G1 phase by using two chemical inhibitors: wortmannin (an inhibitor of PI3K activity), and PD 098059 (an inhibitor of MEK activity). HeLa-neo and HeLa-EJ cells were arrested at mitosis and immediately after their release were treated with different concentrations of wortmannin. After 14 h of being released from the mitotic arrest, most of the wortmanninuntreated HeLa-EJ cells were in S phase (69.2%), while HeLa-neo cells were delayed in G1 phase (63.7%) (Table 1).
Hours after arrest 10
12
14
16
Hours after arrest 10
12
14
16
*
*
**
BrdU incorporation (%)
100
*
**
75
50
** 25
0
Hela-neo
HeLa-EJ
Figure 1. Overexpression of HRAS reduces the G1 phase in HeLa cells. Cell cultures were synchronized in mitosis (as discussed in Materials and Methods) and cell cycle distribution was determined by flow cytometry analysis at different times after release from the arrest. (*p ⬍0.05; **p ⬍0.01). A representative assay of at least three independent experiments is shown.
hours H-ras
0
2
+
4
+
6
+
+
Figure 3. HeLa-EJ cells initiate earlier DNA replication. HeLa-neo and HeLa-EJ cells were treated with 2 mM thymidine for 24 h. After removing the thymidine, 10 µM BrdU was added and its incorporation was determined by flow cytometry at the indicated times. Data represent the mean value ⫾ SD (*p ⬍0.05; **p ⬍0.01).
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
314
Table 1. HRAS effect on HeLa cell cycle is dependent on PI3K/AKT signaling pathway Cell treatment Wortmannin (nM) 0 100 300
G1 HeLa-neo 63.7 ⫾ 1.8 70.3 ⫾ 5.6 70.2 ⫾ 2.9
S HeLa-EJ 22.9 ⫾ 4.3a 54 ⫾ 3.3 62.9 ⫾ 1.2
HeLa-neo 21.9 ⫾ 2.1 14.7 ⫾ 3.9 13.1 ⫾ 3.9
G2/M HeLa-EJ 69.2 ⫾ 3.3 27.6 ⫾ 1.2 61.6 ⫾ 5.1
HeLa-neo 14.4 ⫾ 3.8 14.9 ⫾ 2.7 16.8 ⫾ 3.0
HeLa-EJ 15 ⫾ 3.2 18.4 ⫾ 3.4 20.9 ⫾ 3.9
Note: HeLa-neo and HeLa-EJ cells released from a mitotic arrest were incubated for 14 h in the presence of 0, 0.1 and 0.3 µM of wortmannin. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data are mean ⫾ SD (n ⫽ 3). a p ⬍0.01.
However, wortmannin inhibited, in a dose-dependent manner, the progression from G1 to S phase in both HeLa-neo and HeLa-EJ cells. Although HeLa-EJ cells were more resistant than HeLa-neo to the inhibition of G1/S progression in the presence of wortmannin, their transition to the S phase was significantly blocked at the highest doses of wortmannin (Table 1). On the other hand, we determined the effect of different concentrations of PD 098059 on the HRAS-mediated G1 phase reduction. Similar to the effect observed with wortmannin treatment, the addition of PD 098059 immediately after the release of the cell cultures from the mitotic arrest and during a period of 16 h reduced the progression from G1 to S phase, in a dose-dependent manner, in both HeLa-neo and HeLa-EJ cells, even though HeLa-neo cells were more sensitive to PD 098059 inhibition than HeLa-EJ cells (Table 2). However, the concentration of PD 098059 necessary to significantly inhibit the G1/S progression in HeLa-EJ and HeLa-neo cells was higher than that previously reported for HeLa cells (100 µM) (21). Thus, we evaluated the phosphorylation level of ERK in synchronized cell cultures after treatment with different concentrations of PD 098059. We observed that untreated HeLa-EJ cells had higher levels of phosphorylated ERK than HeLa-neo cells in synchronic cultures released from a mitotic arrest, without affecting protein levels of ERK (Figure 4, lanes 1–2). Using 100 µM of the inhibitor (concentration that does not affect G1/S progression), the phosphorylation level of ERK in HeLa-EJ and HeLa-neo was barely affected (Figure 4, lanes 3–4). However, when we used a concentration of PD 098059 that significantly inhibited G1/S progression (300 µM), the ERK phosphorylation was totally inhibited in both HeLa-neo and
HeLa-EJ (Figure 4, lanes 5–6). Thus, the complete abrogation of MEK activity correlated with a significant inhibition of G1/S transition. Discussion Although HPV infection is the main factor associated with cervical carcinoma, additional factors are needed for the development of cervical cancer. The RAS oncogene is one of the most important genes involved in cancer, and its mutation has been reported in association with HPV infection (7,8). Interestingly, in an HPV-containing cell line (HeLa) we found that overexpression of HRAS oncogene causes a shortening of G1 phase duration and a faster progression from G1/S phase. Previous reports have demonstrated that HRAS oncogene induces cell cycle progression through different signaling pathways. For instance, RAS oncogene induced G1/S progression in NIH 3T3 fibroblasts through the up-regulation of cyclins D and E, by using the Raf, PI3K and RAL pathways (22). In addition, in a human breast carcinoma-derived cell line (MDA468), RAS is able to downregulate the expression of p27, an important inhibitor of the G1/S transition, by means of PI3K and Rho pathways (23). Importantly, a Ras mutant protein able to activate MEK pathway but not PI3K, or another mutant protein able to activate PI3K pathway but not MEK, was defective in producing cell cycle progression. However, the simultaneous overexpression of these two mutants induced the G1/S transition, suggesting that both PI3K and MEK activities are necessary for S phase entry (24). Thus, RAS effects on cell cycle progression seem to depend on different signaling pathways.
Table 2. Inhibition of the RAF/MEK/ERK pathway abrogates the HRAS-induced G1 reduction Cell treatment PD 098059 (µM) 0 100 300
G1 HeLa-neo 34.86 ⫾ 4.6 48.24 ⫾ 1.2 67.57 ⫾ 3.7
S HeLa-EJ 13.7 ⫾ 3.0a 18.04 ⫾ 4.0a 62.09 ⫾ 4.0
HeLa-neo 56.69 ⫾ 5.0 37.25 ⫾ 3.9 16.38 ⫾ 1.0
G2/M HeLa-EJ 37.02 ⫾ 4.0a 47.97 ⫾ 1.0 22.28 ⫾ 3.0
HeLa-neo 8.45 ⫾ 2.0 14.52 ⫾ 3.0 16.05 ⫾ 4.0
HeLa-EJ 49.28 ⫾ 1.0b 33.98 ⫾ 4.0a 15.63 ⫾ 1.2
Note: HeLa-neo and HeLa-EJ cells released from a mitotic arrest were incubated for 16 h in the presence of 0, 100 and 300 µM of PD 098059. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data are mean ⫾ SD (n ⫽ 3). a p ⬍0.05; bp ⬍0.01.
HRAS Induces G1/S Progression in HeLa Cells
1
2
3
4
5
6
pERK
315
Dr. Francisco Vela´zquez and Dr. Humberto Lanz for the critical reading of the manuscript. This work was partially supported by grants from CONACyT [28780N, 38516N (EG) and 25299M (AGC)].
References ERK
Ras µM) PD (µ
+
-
-
+ 100
-
+ 300
Figure 4. Effect of PD 098059 on ERK phosphorylation. HeLa cells released from a mitotic arrest were incubated with PD 098059 for 16 h. Whole cell lysates were obtained and analyzed by Western blot, using anti-phosphospecific ERK antibodies. Membranes were stripped and re-blotted with anti-ERK antibodies. Lanes 1, 3 and 5: HeLa-neo cells. Lanes 2, 4 and 6: HeLa-EJ cells. Lanes 1 and 2: untreated cells. Lanes 2 and 4: PD 098059 100 µM. Lanes 5 and 6: PD 098059 300 µM. ERK ⫽ total ERK; pERK ⫽ phosphorylated ERK. A representative assay of two independent experiments is shown.
As a first attempt to elucidate the pathway used by HRAS to induce the effects observed on cell cycle progression, we inhibited two important effectors of RAS signaling, MEK and PI3K, using specific inhibitors (PD 098059 and wortmannin, respectively). We determined that the inhibition of either PI3K or MEK is sufficient to abrogate the G1 phase reduction mediated by HRAS oncogene (Tables 1 and 2, respectively). These data suggest that HRAS is able to further affect cell cycle progression in the presence of HPV through both the PI3K and MEK routes. However, there is also the possibility of a cross-talk between PI3K and MEK proteins where the inhibition of one of these pathways results in the inactivation of the other one. Although several reports have described a cross-talk between the PI3K/AKT and RAF/MEK/ERK pathways, the functional interaction of both pathways seems to be celltype dependent (25–27). However, this cross-talk has not been described specifically in HeLa cells yet, but it has been reported that the inhibition of PI3K has no effect on MEK activity (28,29). Based on these observations, we think that the abrogation of the HRAS-induced reduction of G1 phase by the inhibition of either MEK or PI3K pathways is not a consequence of a cross-talk but a synergism between them. For instance, both PI3K and MEK pathways are required for cyclin D protein expression and stability, as well as downregulation of the cdk inhibitor p27 (30–32). Our results suggest that mutational activation of RAS genes could play an important role in cervical cancer development.
Acknowledgments We gratefully acknowledge Blanca Reyes, Pedro Chavez and Miriam C. Guido for helpful technical assistance. We also thank
1. Einstein MH, Goldberg GL. Human papillomavirus and cervical neoplasia. Cancer Invest 2002;20:1080–1085. 2. Lizano M, Berumen J, Guido MC, Casas L, Garcia-Carranca A. Association between human papillomavirus type 18 variants and histopathology of cervical cancer. J Natl Cancer Inst 1997;89:1227–1231. 3. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–19. 4. Furumoto H, Irahara M. Human papillomavirus (HPV) and cervical cancer. J Med Invest 2002;49:124–133. 5. Bos JL. ras oncogenes in human cancer. Cancer Res 1989;49: 4682–4689. 6. Rodenhuis S. Ras and human tumors. Semin Cancer Biol 1992;3: 241–247. 7. Stenzel A, Semczuk A, Rozynskal K, Jakowicki J, Wojcierowski J. Lowrisk and high-risk HPV-infection and K-ras gene point mutations in human cervical cancer: a study of 31 cases. Pathol Res Pract 2001; 197:597–603. 8. Alonio LV, Picconi MA, Dalbert D, Mural J, Bartt O, Bazan G, Dominguez M, Teyssie AR. Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix. J Clin Virol 2003;27:263–269. 9. Ayllon V, Rebollo A. Ras-induced cellular events. Mol Membr Biol 2000;17:65–73. 10. Moodie SA, Willumsen BM, Weber MJ, Wolfman A. Complexes of Ras GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 1993;260:1658–1661. 11. Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 1994;370:527–532. 12. Winston JT, Coats SR, Wang YZ, Pledger WJ. Regulation of the cell cycle machinery by oncogenic ras. Oncogene 1996;12:127–134. 13. Macaluso M, Russo G, Cinti C, Bazan V, Gebbia N, Russo A. Ras family genes: an interesting link between cell cycle and cancer. J Cell Physiol 2002;192:125–130. 14. Peeper DS, Upton TM, Ladha MH, Neuman E, Zalvide J, Bernards R, DeCaprio JA, Ewen ME. Ras signaling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 1997;386:177–181. 15. Mittnacht S, Paterson H, Olson MF, Marshall CJ. Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein. Curr Biol 1997;7:219–221. 16. Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR. Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 1997;387:422–426. 17. Miranda EI, Santana C, Rojas E, Hernandez S, Ostrosky-Wegman P, Garcia-Carranca A. Induced mitotic death of HeLa cells by abnormal expression of c-H-ras. Mutat Res 1996;349:173–182. 18. Santana C, Ortega E, Garcia-Carranca A. Oncogenic H-ras induces cyclin B expression in a p53-independent manner. Mutat Res 2002; 508:49–58. 19. Palisano JR. The absence of confronting cisternae following thymidineinduced synchronization in HeLa cells. Cell Biol Int 1993;17:653–664. 20. Crespo P, Leon J. Ras proteins in the control of the cell cycle and cell differentiation. Cell Mol Life Sci 2000;57:1613–1636. 21. Warne-Cramer BJ, Cottrel GT, Burt JUM, Lau AF. Regulation of connexin-43 gap junctional intercellular communication by mitogenactivated protein kinase. J Biol Chem 1998;273:9188–9196.
316
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
22. Gille H, Downward J. Multiple ras effectors contribute to G1 cell cycle progression. J Biol Chem 1999;274:22033–22040. 23. Vidal A, Millard SS, Miller JP, Koff A. Rho activity can alter the translation of p27 mRNA and is important for RasV12-induced transformation in a manner dependent on p27 status. J Biol Chem 2002; 277:16433–16440. 24. Yang JJ, Kang JS, Krauss RS. Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth. Mol Cell Biol 1998;18:2586–2595. 25. Chaudhary A, King WG, Mattaliano MD, Frost JA, Diaz B, Morrison DK, Cobb MH, Marshall MS, Brugge JS. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 2000;10:551–554. 26. Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek AB. Negative regulation of the serine/threonine kinase B-Raf by Akt. J Biol Chem 2000;275:27354–27359. 27. Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M. Regulation of Raf-AKT cross-talk. J Biol Chem 2002;277:31099– 31106.
28. Poser S, Impey S, Trinh K, Xia Z, Storm DR. SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation. EMBO J 2000;19:4955–4966. 29. Roberts EC, Shapiro PS, Nahreini TS, Pages G, Pouyssegur J, Ahn NG. Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis. Mol Cell Biol 2002;22:7226–7241. 30. Cheng M, Sexl V, Sherr CJ, Roussel MF. Assembly of cyclin Ddependent kinase and titration of p27KIP1 regulated by mitogen-activated protein kinase kinase (MEK1). Proc Natl Acad Sci 1998;95: 1091–1096. 31. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expression is controlled post-trancriptionally via a phosphatidylinositol 3-kinase/AKT-dependent pathway. J Biol Chem 1998;273:29864–29872. 32. Rivard N, Boucher MJ, Asselin C, L’Allemain G. MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblast and epithelial cells. Am J Physiol 1999;277:C652–C664.
ORIGINAL ARTICLE
Effects of HRAS Oncogene on Cell Cycle Progression in a Cervical Cancer-Derived Cell Line Emilio Co´rdova-Alarco´n,a Federico Centeno,b Jorge Reyes-Esparza,c Alejandro Garcı´a-Carranca´d,e and Efraı´n Garridob a
Departamento de Biomedicina Molecular, bDepartamento de Gene´tica y Biologı´a Molecular, Centro de Investigacio´n y de Estudios Avanzados del Instituto Polite´cnico Nacional, Me´xico, D.F., Me´xico c Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Me´xico d Unidad de Investigacio´n Biome´dica en Ca´ncer, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Me´xico, D.F., Me´xico e Instituto Nacional de Cancerologı´a, Secretarı´a de Salud, Me´xico, D.F., Me´xico Received for publication March 5, 2004; accepted July 12, 2004 (04/043).
Background. Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes. Methods. HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways. Results. We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase. Conclusions. Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells. 쑖 2005 IMSS. Published by Elsevier Inc. Key Words: Cervical carcinoma, HPV, RAS, Flow cytometry.
Introduction
Address reprint requests to: Dr. Efraı´n Garrido, Departamento de Gene´tica y Biologı´a Molecular, CINVESTAV-IPN, Av. IPN No. 2508, Col. San Pedro Zacatenco, 07360 Me´xico D.F., Me´xico. Phone: (⫹52) (55) 50613800 ext. 5373; FAX: (⫹52) (55) 5061-3800 ext. 5372; E-mail: [email protected]
0188-4409/05 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 5 .0 4 .0 01
Cervical carcinoma accounts for about 10% of all newly diagnosed cancers in women worldwide (1) and is one of the most frequent tumors in Mexico (2). Human papillomavirus infection (HPV) is the main factor associated with cervical carcinoma (3). The HPV gene products E6 and E7 play a critical role in cervical carcinogenesis by interfering with
쑖 2005 IMSS. Published by Elsevier Inc.
312
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
p53 and Rb functions, respectively, and deregulating the cell cycle (4). It has been realized that HPV infection is not sufficient for cervical carcinoma development, and that the presence of additional factors is needed (3). RAS proto-oncogenes are among the most frequently mutated genes in different human tumors (5,6). Mutations in RAS genes have been shown to coexist with oncogenic HPV in cervical cancer (7). In addition, RAS mutations are associated with a faster progression of HPV-induced lesions of uterine cervix (8). In mammals there are at least three RAS genes: NRAS, KRAS, and HRAS and the corresponding proteins function as regulators of different proliferation and survival signals (9). Although several putative RAS effectors have been reported, the RAF/MAPK-ERK kinase (MEK)/ extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/AKT pathways have been described as the most important (10,11). Although it has been described that in the absence of a functional Rb protein all the effects of RAS in cell cycle regulation are lost (12,13), recently several reports suggest that RAS have additional functions on cell cycle progression (14–16). In fact, neutralizing antibodies against Ras protein still inhibit DNA replication in Rb knockout cells (14,15). Additionally, it has been shown that MYC and RAS cooperate to induce cell cycle progression into S phase in an Rb-independent manner (16). These observations suggest that RAS oncogene could regulate G1/S progression independently of Rb protein. Therefore, despite the absence of a functional Rb protein in cervical cancer, RAS could participate in the development of this kind of tumor. In this work, we investigated whether the overexpression of oncogenic HRAS could alter cell cycle progression in the HeLa cell line containing the HPV-18 genome. We found that HRAS overexpression reduced G1 phase duration. Moreover, the G1/S transition was faster in overexpressing HRAS HeLa cells. Finally, using RAS-signaling chemical inhibitors, we showed that RAF/MEK/ERK and PI3K/AKT pathways participate in the G1 phase reduction mediated by HRAS.
Materials and Methods Cell culture. HeLa cells were stably transfected with plasmid pSV2neo HRAS Val12 (HeLa-EJ) or pSV2neo empty vector (HeLa-neo) and have already been described (17,18). Cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM) and 10% newborn calf serum at 37⬚C in 5% CO2 atmosphere. Experiments were performed on subconfluent cultures not less than 24 h after plating. Drug treatments. The different treatments were performed at a cell density of 3.5 × 105 cells per 60-mm plate. Thymidine [50-89-5] (Amersham, Piscataway, NJ) was used at
2 mM (final concentration) for 24 h, and the cells were trypsinized at different times post-treatment. For mitotic arrest, cells were incubated for 24 h with 2 mM thymidine, washed and nocodazole [31430–18–9] (Sigma, St. Louis, MO) was added at 0.1 µg/mL for 15 h. Floating cells from the supernatant (mitotic cells) were recovered, washed out extensively, re-seeded and harvested at different time intervals. HeLa-EJ and neo cells were synchronized at mitosis as described above. After releasing them from mitotic arrest, different concentrations of the chemical inhibitors PD 098059 [167869–21–8] (Sigma) or wortmannin [19545–26– 7] (Sigma) were added and cells harvested and analyzed by flow cytometry. Flow cytometry analysis. One million cells treated or not with the different drugs were pelleted by low-speed centrifugation and the pellet resuspended with PBS. One mL of icecold 80% ethanol was added and the cell suspension was incubated overnight at 4⬚C. Fixed cells were harvested by centrifugation, resuspended in PBS containing RNase A (100 µg/mL) and propidium iodide (50 µg/mL), then incubated in the dark at 4⬚C for 1 h. The ethanol-fixed and propidium iodide-stained cells were analyzed for their DNA content in a FACS sort flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) (BDIS). Data analysis was performed using CellQuest (BDIS) and Modfit software (Verity Software House, Topsham, ME). Cell lysates and Western blot. Cells were harvested by trypsinization, washed with PBS and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4) supplemented with a mixture of phosphatase and protease inhibitors (1 mM sodium orthovanadate and 2 mM sodium fluoride, 1 mM PMSF, aprotinin 100 µg/mL, leupeptin 100 µg/mL, pepstatin 10 µg/mL, benzamidin 500 µg/mL, antipain 50 µg/mL and chymostatin 50 µg/mL). After incubation for 30 min on ice, lysates were cleared by centrifugation at 10,000 rpm for 10 min and protein concentration quantified with BioRad DC protein assay (BioRad, Hercules, CA). Equal amounts of proteins from the different samples were electrophoretically separated on a denaturating polyacrylamide gel (SDS-PAGE). Proteins were transferred to Hybond membrane (Amersham) and blocked with 5% non-fat milk in PBS. Membranes were incubated with anti-phosphospecific ERK antibodies (Cell Signaling, Beverly, MA), followed by horseradish peroxidase-conjugated goat anti-rabbit antibody (Zymed, South San Francisco, CA). Proteins were observed using the ECL detection system (Amersham) followed by exposure to XOMAT films (Kodak, Rochester, NY). Membranes were stripped and re-blotted with anti-ERK antibodies (Cell Signaling). Statistical analysis. The statistical significance of the difference between intergroup comparisons was obtained using
HRAS Induces G1/S Progression in HeLa Cells
Student’s t-test. Data were expressed as mean ⫾ SD and were representative of at least three independent experiments.
Expression of exogenous HRAS results in the shortening of G1 phase duration in an HPV expressing cell line. In order to evaluate if HRAS oncogene can further affect cell cycle progression in the presence of HPV, we synchronized HeLa cells (a cell line derived from a human cervical carcinoma that expresses E6 and E7 from HPV-18), stably transfected with HRAS oncogene (HeLa-EJ) or empty vector (HeLa-neo). Initially, cell cultures were synchronized in mitosis (by thymidine–nocodazole treatment) and cell percentage in G1 phase was determined several hours after releasing the cells from the arrest. During the first 10 h after the release from the mitotic arrest, most of the HeLa-neo and HeLa-EJ cells were in G1 phase (⬎90%). However, after 12 h, the percentage of HeLa-EJ cells in G1 phase was consistently reduced in comparison with HeLa-neo cells. About 16 h later, while HeLa-neo cells were at the beginning of S phase, HeLa-EJ cells were already at the end of the same phase (Figure 1). Cell cycle reduction induced by HRAS occurred in late G1. An excess of thymidine is able to induce a reversible cell cycle arrest in G1/S in cell lines from different origins (including HeLa) (19). Incubation of cell cultures with 2 mM thymidine for 24 h induced a cell cycle arrest in G1/S in both HeLa-neo and HeLa-EJ cells (Figure 2). However, 2 h after thymidine was removed from the cultures, more than 80% of HeLa-EJ cells were in S phase. A similar percentage of HeLa-neo cells were in S phase only 6 h after removing the thymidine (Figure 2). BrdU incorporation assays were performed in order to determine DNA synthesis rate after thymidine arrest. These assays confirmed that DNA synthesis began in HeLa-EJ cells only 2 h after thymidine removal, whereas in HeLa-neo cells DNA synthesis began 4 h after thymidine was removed (Figure 3).
100
Percentage of cells in S
Results
313
**
**
75
50
25
0 0
2
4
6
8
10
12
hours Figure 2. HeLa cells stably transfected with HRAS show a faster G1/S progression. Cell cultures were incubated with 2 mM thymidine for 24 h and DNA content was determined several hours post-treatment by flow cytometry. Time 0 corresponds to 24 h of incubation with 2 mM thymidine. Data represent the mean value ⫾ SD. (**p ⬍0.01). HeLa-neo (䊐), HeLaEJ ( ).
The shortening of G1 phase induced by HRAS is dependent on RAF/MEK/ERK and PI3K/AKT pathways. The RAF/ MEK/ERK or PI3K/AKT pathways mediate the expression effects of HRAS on cell cycle regulation in different cell types (20). Therefore, we assessed the implication of these pathways in the HRAS-dependent reduction of G1 phase by using two chemical inhibitors: wortmannin (an inhibitor of PI3K activity), and PD 098059 (an inhibitor of MEK activity). HeLa-neo and HeLa-EJ cells were arrested at mitosis and immediately after their release were treated with different concentrations of wortmannin. After 14 h of being released from the mitotic arrest, most of the wortmanninuntreated HeLa-EJ cells were in S phase (69.2%), while HeLa-neo cells were delayed in G1 phase (63.7%) (Table 1).
Hours after arrest 10
12
14
16
Hours after arrest 10
12
14
16
*
*
**
BrdU incorporation (%)
100
*
**
75
50
** 25
0
Hela-neo
HeLa-EJ
Figure 1. Overexpression of HRAS reduces the G1 phase in HeLa cells. Cell cultures were synchronized in mitosis (as discussed in Materials and Methods) and cell cycle distribution was determined by flow cytometry analysis at different times after release from the arrest. (*p ⬍0.05; **p ⬍0.01). A representative assay of at least three independent experiments is shown.
hours H-ras
0
2
+
4
+
6
+
+
Figure 3. HeLa-EJ cells initiate earlier DNA replication. HeLa-neo and HeLa-EJ cells were treated with 2 mM thymidine for 24 h. After removing the thymidine, 10 µM BrdU was added and its incorporation was determined by flow cytometry at the indicated times. Data represent the mean value ⫾ SD (*p ⬍0.05; **p ⬍0.01).
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
314
Table 1. HRAS effect on HeLa cell cycle is dependent on PI3K/AKT signaling pathway Cell treatment Wortmannin (nM) 0 100 300
G1 HeLa-neo 63.7 ⫾ 1.8 70.3 ⫾ 5.6 70.2 ⫾ 2.9
S HeLa-EJ 22.9 ⫾ 4.3a 54 ⫾ 3.3 62.9 ⫾ 1.2
HeLa-neo 21.9 ⫾ 2.1 14.7 ⫾ 3.9 13.1 ⫾ 3.9
G2/M HeLa-EJ 69.2 ⫾ 3.3 27.6 ⫾ 1.2 61.6 ⫾ 5.1
HeLa-neo 14.4 ⫾ 3.8 14.9 ⫾ 2.7 16.8 ⫾ 3.0
HeLa-EJ 15 ⫾ 3.2 18.4 ⫾ 3.4 20.9 ⫾ 3.9
Note: HeLa-neo and HeLa-EJ cells released from a mitotic arrest were incubated for 14 h in the presence of 0, 0.1 and 0.3 µM of wortmannin. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data are mean ⫾ SD (n ⫽ 3). a p ⬍0.01.
However, wortmannin inhibited, in a dose-dependent manner, the progression from G1 to S phase in both HeLa-neo and HeLa-EJ cells. Although HeLa-EJ cells were more resistant than HeLa-neo to the inhibition of G1/S progression in the presence of wortmannin, their transition to the S phase was significantly blocked at the highest doses of wortmannin (Table 1). On the other hand, we determined the effect of different concentrations of PD 098059 on the HRAS-mediated G1 phase reduction. Similar to the effect observed with wortmannin treatment, the addition of PD 098059 immediately after the release of the cell cultures from the mitotic arrest and during a period of 16 h reduced the progression from G1 to S phase, in a dose-dependent manner, in both HeLa-neo and HeLa-EJ cells, even though HeLa-neo cells were more sensitive to PD 098059 inhibition than HeLa-EJ cells (Table 2). However, the concentration of PD 098059 necessary to significantly inhibit the G1/S progression in HeLa-EJ and HeLa-neo cells was higher than that previously reported for HeLa cells (100 µM) (21). Thus, we evaluated the phosphorylation level of ERK in synchronized cell cultures after treatment with different concentrations of PD 098059. We observed that untreated HeLa-EJ cells had higher levels of phosphorylated ERK than HeLa-neo cells in synchronic cultures released from a mitotic arrest, without affecting protein levels of ERK (Figure 4, lanes 1–2). Using 100 µM of the inhibitor (concentration that does not affect G1/S progression), the phosphorylation level of ERK in HeLa-EJ and HeLa-neo was barely affected (Figure 4, lanes 3–4). However, when we used a concentration of PD 098059 that significantly inhibited G1/S progression (300 µM), the ERK phosphorylation was totally inhibited in both HeLa-neo and
HeLa-EJ (Figure 4, lanes 5–6). Thus, the complete abrogation of MEK activity correlated with a significant inhibition of G1/S transition. Discussion Although HPV infection is the main factor associated with cervical carcinoma, additional factors are needed for the development of cervical cancer. The RAS oncogene is one of the most important genes involved in cancer, and its mutation has been reported in association with HPV infection (7,8). Interestingly, in an HPV-containing cell line (HeLa) we found that overexpression of HRAS oncogene causes a shortening of G1 phase duration and a faster progression from G1/S phase. Previous reports have demonstrated that HRAS oncogene induces cell cycle progression through different signaling pathways. For instance, RAS oncogene induced G1/S progression in NIH 3T3 fibroblasts through the up-regulation of cyclins D and E, by using the Raf, PI3K and RAL pathways (22). In addition, in a human breast carcinoma-derived cell line (MDA468), RAS is able to downregulate the expression of p27, an important inhibitor of the G1/S transition, by means of PI3K and Rho pathways (23). Importantly, a Ras mutant protein able to activate MEK pathway but not PI3K, or another mutant protein able to activate PI3K pathway but not MEK, was defective in producing cell cycle progression. However, the simultaneous overexpression of these two mutants induced the G1/S transition, suggesting that both PI3K and MEK activities are necessary for S phase entry (24). Thus, RAS effects on cell cycle progression seem to depend on different signaling pathways.
Table 2. Inhibition of the RAF/MEK/ERK pathway abrogates the HRAS-induced G1 reduction Cell treatment PD 098059 (µM) 0 100 300
G1 HeLa-neo 34.86 ⫾ 4.6 48.24 ⫾ 1.2 67.57 ⫾ 3.7
S HeLa-EJ 13.7 ⫾ 3.0a 18.04 ⫾ 4.0a 62.09 ⫾ 4.0
HeLa-neo 56.69 ⫾ 5.0 37.25 ⫾ 3.9 16.38 ⫾ 1.0
G2/M HeLa-EJ 37.02 ⫾ 4.0a 47.97 ⫾ 1.0 22.28 ⫾ 3.0
HeLa-neo 8.45 ⫾ 2.0 14.52 ⫾ 3.0 16.05 ⫾ 4.0
HeLa-EJ 49.28 ⫾ 1.0b 33.98 ⫾ 4.0a 15.63 ⫾ 1.2
Note: HeLa-neo and HeLa-EJ cells released from a mitotic arrest were incubated for 16 h in the presence of 0, 100 and 300 µM of PD 098059. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data are mean ⫾ SD (n ⫽ 3). a p ⬍0.05; bp ⬍0.01.
HRAS Induces G1/S Progression in HeLa Cells
1
2
3
4
5
6
pERK
315
Dr. Francisco Vela´zquez and Dr. Humberto Lanz for the critical reading of the manuscript. This work was partially supported by grants from CONACyT [28780N, 38516N (EG) and 25299M (AGC)].
References ERK
Ras µM) PD (µ
+
-
-
+ 100
-
+ 300
Figure 4. Effect of PD 098059 on ERK phosphorylation. HeLa cells released from a mitotic arrest were incubated with PD 098059 for 16 h. Whole cell lysates were obtained and analyzed by Western blot, using anti-phosphospecific ERK antibodies. Membranes were stripped and re-blotted with anti-ERK antibodies. Lanes 1, 3 and 5: HeLa-neo cells. Lanes 2, 4 and 6: HeLa-EJ cells. Lanes 1 and 2: untreated cells. Lanes 2 and 4: PD 098059 100 µM. Lanes 5 and 6: PD 098059 300 µM. ERK ⫽ total ERK; pERK ⫽ phosphorylated ERK. A representative assay of two independent experiments is shown.
As a first attempt to elucidate the pathway used by HRAS to induce the effects observed on cell cycle progression, we inhibited two important effectors of RAS signaling, MEK and PI3K, using specific inhibitors (PD 098059 and wortmannin, respectively). We determined that the inhibition of either PI3K or MEK is sufficient to abrogate the G1 phase reduction mediated by HRAS oncogene (Tables 1 and 2, respectively). These data suggest that HRAS is able to further affect cell cycle progression in the presence of HPV through both the PI3K and MEK routes. However, there is also the possibility of a cross-talk between PI3K and MEK proteins where the inhibition of one of these pathways results in the inactivation of the other one. Although several reports have described a cross-talk between the PI3K/AKT and RAF/MEK/ERK pathways, the functional interaction of both pathways seems to be celltype dependent (25–27). However, this cross-talk has not been described specifically in HeLa cells yet, but it has been reported that the inhibition of PI3K has no effect on MEK activity (28,29). Based on these observations, we think that the abrogation of the HRAS-induced reduction of G1 phase by the inhibition of either MEK or PI3K pathways is not a consequence of a cross-talk but a synergism between them. For instance, both PI3K and MEK pathways are required for cyclin D protein expression and stability, as well as downregulation of the cdk inhibitor p27 (30–32). Our results suggest that mutational activation of RAS genes could play an important role in cervical cancer development.
Acknowledgments We gratefully acknowledge Blanca Reyes, Pedro Chavez and Miriam C. Guido for helpful technical assistance. We also thank
1. Einstein MH, Goldberg GL. Human papillomavirus and cervical neoplasia. Cancer Invest 2002;20:1080–1085. 2. Lizano M, Berumen J, Guido MC, Casas L, Garcia-Carranca A. Association between human papillomavirus type 18 variants and histopathology of cervical cancer. J Natl Cancer Inst 1997;89:1227–1231. 3. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–19. 4. Furumoto H, Irahara M. Human papillomavirus (HPV) and cervical cancer. J Med Invest 2002;49:124–133. 5. Bos JL. ras oncogenes in human cancer. Cancer Res 1989;49: 4682–4689. 6. Rodenhuis S. Ras and human tumors. Semin Cancer Biol 1992;3: 241–247. 7. Stenzel A, Semczuk A, Rozynskal K, Jakowicki J, Wojcierowski J. Lowrisk and high-risk HPV-infection and K-ras gene point mutations in human cervical cancer: a study of 31 cases. Pathol Res Pract 2001; 197:597–603. 8. Alonio LV, Picconi MA, Dalbert D, Mural J, Bartt O, Bazan G, Dominguez M, Teyssie AR. Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix. J Clin Virol 2003;27:263–269. 9. Ayllon V, Rebollo A. Ras-induced cellular events. Mol Membr Biol 2000;17:65–73. 10. Moodie SA, Willumsen BM, Weber MJ, Wolfman A. Complexes of Ras GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 1993;260:1658–1661. 11. Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 1994;370:527–532. 12. Winston JT, Coats SR, Wang YZ, Pledger WJ. Regulation of the cell cycle machinery by oncogenic ras. Oncogene 1996;12:127–134. 13. Macaluso M, Russo G, Cinti C, Bazan V, Gebbia N, Russo A. Ras family genes: an interesting link between cell cycle and cancer. J Cell Physiol 2002;192:125–130. 14. Peeper DS, Upton TM, Ladha MH, Neuman E, Zalvide J, Bernards R, DeCaprio JA, Ewen ME. Ras signaling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 1997;386:177–181. 15. Mittnacht S, Paterson H, Olson MF, Marshall CJ. Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein. Curr Biol 1997;7:219–221. 16. Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR. Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 1997;387:422–426. 17. Miranda EI, Santana C, Rojas E, Hernandez S, Ostrosky-Wegman P, Garcia-Carranca A. Induced mitotic death of HeLa cells by abnormal expression of c-H-ras. Mutat Res 1996;349:173–182. 18. Santana C, Ortega E, Garcia-Carranca A. Oncogenic H-ras induces cyclin B expression in a p53-independent manner. Mutat Res 2002; 508:49–58. 19. Palisano JR. The absence of confronting cisternae following thymidineinduced synchronization in HeLa cells. Cell Biol Int 1993;17:653–664. 20. Crespo P, Leon J. Ras proteins in the control of the cell cycle and cell differentiation. Cell Mol Life Sci 2000;57:1613–1636. 21. Warne-Cramer BJ, Cottrel GT, Burt JUM, Lau AF. Regulation of connexin-43 gap junctional intercellular communication by mitogenactivated protein kinase. J Biol Chem 1998;273:9188–9196.
316
Co´rdova-Alarco´n et al. / Archives of Medical Research 36 (2005) 311–316
22. Gille H, Downward J. Multiple ras effectors contribute to G1 cell cycle progression. J Biol Chem 1999;274:22033–22040. 23. Vidal A, Millard SS, Miller JP, Koff A. Rho activity can alter the translation of p27 mRNA and is important for RasV12-induced transformation in a manner dependent on p27 status. J Biol Chem 2002; 277:16433–16440. 24. Yang JJ, Kang JS, Krauss RS. Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth. Mol Cell Biol 1998;18:2586–2595. 25. Chaudhary A, King WG, Mattaliano MD, Frost JA, Diaz B, Morrison DK, Cobb MH, Marshall MS, Brugge JS. Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338. Curr Biol 2000;10:551–554. 26. Guan KL, Figueroa C, Brtva TR, Zhu T, Taylor J, Barber TD, Vojtek AB. Negative regulation of the serine/threonine kinase B-Raf by Akt. J Biol Chem 2000;275:27354–27359. 27. Moelling K, Schad K, Bosse M, Zimmermann S, Schweneker M. Regulation of Raf-AKT cross-talk. J Biol Chem 2002;277:31099– 31106.
28. Poser S, Impey S, Trinh K, Xia Z, Storm DR. SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation. EMBO J 2000;19:4955–4966. 29. Roberts EC, Shapiro PS, Nahreini TS, Pages G, Pouyssegur J, Ahn NG. Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis. Mol Cell Biol 2002;22:7226–7241. 30. Cheng M, Sexl V, Sherr CJ, Roussel MF. Assembly of cyclin Ddependent kinase and titration of p27KIP1 regulated by mitogen-activated protein kinase kinase (MEK1). Proc Natl Acad Sci 1998;95: 1091–1096. 31. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expression is controlled post-trancriptionally via a phosphatidylinositol 3-kinase/AKT-dependent pathway. J Biol Chem 1998;273:29864–29872. 32. Rivard N, Boucher MJ, Asselin C, L’Allemain G. MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblast and epithelial cells. Am J Physiol 1999;277:C652–C664.