Docsity
Docsity

Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity


Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan


Guidelines and tips
Guidelines and tips

Vitamin C & P53: Enhancing Chemo Response in Cervical Cancer - Prof. Jessica L. Tischler, Study notes of Organic Chemistry

The role of vitamin c in enhancing the response of cervical carcinoma hela cells to chemotherapeutic drugs by stabilizing the p53 protein. The study suggests that vitamin c downregulates the viral oncoprotein e6, decreases ap-1 members c-jun and c-fos, and upregulates p53, bax, and downregulates bcl2. The findings have potential clinical relevance as they indicate that vitamin c can increase the susceptibility of cervical carcinoma cells to chemotherapeutic drugs.

Typology: Study notes

2012/2013

Uploaded on 09/10/2013

angelo-de-la-casa
angelo-de-la-casa 🇺🇸

4.4

(56)

508 documents

1 / 7

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
Vitamin C Augments Chemotherapeutic Response of
Cervical Carcinoma HeLa Cells by Stabilizing P53
Vijay G. Reddy, Neeru Khanna, and Neeta Singh1
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Received February 20, 2001
Human Papilloma Virus (HPV) is associated in most
instances with cervical cancer. The HPV oncoproteins
target P53 protein for degradation, leading to deregu-
lation of cell cycle. We investigated whether stabiliza-
tion of P53 in cervical cancer cells, by downregulating
HPV transcription would restore the apoptotic ability
of these cells. Our findings show that vitamin C down-
regulates the redox sensitive transcription factor AP-1
and decreases one of its transcription targets HPV E6,
and stabilizes P53. This was associated with an in-
crease in Bax and decrease in Bcl-2 and telomerase
activity. Accumulation of P53 and its target gene bax
then sensitized HeLa cells to cell-cycle arrest, cell
death/apoptosis induced by cisplatin, and etoposide.
Increasing drug sensitivity of cervical carcinoma cells
by stabilizing P53 using vitamin C is a novel approach
and has potential clinical relevance. © 2001 Academic Press
Key Words: vitamin C; cervical cancer; P53, HPV; E6;
chemotherapy.
Cervical cancer has a high prevalence especially
amongst women in Asia and is a leading cause of can-
cer death. It primarily has a viral etiology and HPVs
have been shown to be involved in the pathogenesis of
cervical, vulval, penile, and perianal cancer (1). The
oncogenic potential of high risk HPV types 16, 18, and
to some extent types 33, 45, 52, 58 is attributed to E6
and E7 oncogene products, which participate in trans-
forming the infected cell. The E6 oncoprotein targets
P53 protein, induces its ubiquitin-mediated destruc-
tion (2) and affects its cell-cycle regulation. E6 and E7
oncoproteins are also involved in the activation of te-
lomerase, which contributes to the immortalization of
transformed cells (3). It is shown that absence of P53
leads to abolition of G1 arrest or apoptosis in response
to ionizing radiation and DNA damaging agents (4).
Some reports have associated loss of P53, with in-
creased sensitivity to chemotherapy, whereas others to
increased chemoresistance (46). Cervical cancer che-
motherapy in vivo improved in cases with high P53
expression in the tumor tissue (7, 8). It has been shown
that transactivation and DNA-binding affinity of acti-
vator protein (AP-1) as well as P53 can be modulated
by both posttranslation modifications as well as by
alteration of intracellular redox state (9, 10). Cervical
cancer is associated with low antioxidant status (11).
Vitamin C has been suggested to play a protective role
in development of cervical intraepithelial neoplasia
(CIN) (12), however, its role in the treatment of cervical
cancer has not been studied.
To investigate the effect of vitamin C on the tran-
scriptional regulation of HPV E6/E7 oncogene expres-
sion, HPV-18 positive Hela cells were treated with
noncytotoxic amounts of vitamin C. The findings sug-
gest that vitamin C caused downregulation of the viral
oncoprotein E6, which was paralleled by a decrease of
AP-1 members c-jun and c-fos in a dose- and time-
dependent manner. The downregulation of E6 resulted
in the up regulation of proapoptotic, P53 and Bax pro-
teins but downregulation of apoptotic inhibitor Bcl-2.
There was also significant decrease in telomerase ac-
tivity. Vitamin C also increased the susceptibility/
apoptosis induced by cisplatin and etoposide.
MATERIALS AND METHODS
Cell culture. The human cervical carcinoma cell line (Hela) was
obtained from National Centre for Cell Science, Pune, India and
maintained in DMEM medium containing 10% fetal calf serum and
antibiotics in a humidified atmosphere of 5% CO2in air at 37°C.
Logarithmically growing cells were used for all experiments.
Drug treatment and cell viability assay. 1104cells seeded in
96-well microtiter plates were treated with in vivo achievable con-
centrations of vitamin C ranging from 0.1
M to 10 mM and/or
chemotherapeutic drugs i.e., cisplatin (2–100
M), etoposide (2–100
M), adriamycin (0.01–10
M), bleomycin (0.0004– 0.4 U/ml). The
growth inhibitory effect of vitamin C and chemotherapeutic drugs
was assessed by the MTT assay. After 48 h of incubation, 100
lof5
mg/ml of MTT was added followed by incubation for4hat37°C. The
1To whom correspondence should be addressed at Department of
Biochemistry, Room No 3027-A, All India Institute of Medical Sci-
ences, Ansari Nagar, New Delhi-110029, India. Fax: 91-11-6862663.
E-mail: singh_neeta@hotmail.com.
Biochemical and Biophysical Research Communications 282, 409– 415 (2001)
doi:10.1006/bbrc.2001.4593, available online at http://www.idealibrary.com on
409 0006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
pf3
pf4
pf5

Partial preview of the text

Download Vitamin C & P53: Enhancing Chemo Response in Cervical Cancer - Prof. Jessica L. Tischler and more Study notes Organic Chemistry in PDF only on Docsity!

Vitamin C Augments Chemotherapeutic Response of

Cervical Carcinoma HeLa Cells by Stabilizing P

Vijay G. Reddy, Neeru Khanna, and Neeta Singh

1

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

Received February 20, 2001

Human Papilloma Virus (HPV) is associated in most

instances with cervical cancer. The HPV oncoproteins

target P53 protein for degradation, leading to deregu-

lation of cell cycle. We investigated whether stabiliza-

tion of P53 in cervical cancer cells, by downregulating

HPV transcription would restore the apoptotic ability

of these cells. Our findings show that vitamin C down-

regulates the redox sensitive transcription factor AP-

and decreases one of its transcription targets HPV E6,

and stabilizes P53. This was associated with an in-

crease in Bax and decrease in Bcl-2 and telomerase

activity. Accumulation of P53 and its target gene bax

then sensitized HeLa cells to cell-cycle arrest, cell

death/apoptosis induced by cisplatin, and etoposide.

Increasing drug sensitivity of cervical carcinoma cells

by stabilizing P53 using vitamin C is a novel approach

and has potential clinical relevance. © 2001 Academic Press

Key Words: vitamin C; cervical cancer; P53, HPV; E6;

chemotherapy.

Cervical cancer has a high prevalence especially

amongst women in Asia and is a leading cause of can- cer death. It primarily has a viral etiology and HPVs

have been shown to be involved in the pathogenesis of cervical, vulval, penile, and perianal cancer (1). The

oncogenic potential of high risk HPV types 16, 18, and

to some extent types 33, 45, 52, 58 is attributed to E and E7 oncogene products, which participate in trans-

forming the infected cell. The E6 oncoprotein targets P53 protein, induces its ubiquitin-mediated destruc-

tion (2) and affects its cell-cycle regulation. E6 and E oncoproteins are also involved in the activation of te-

lomerase, which contributes to the immortalization of transformed cells (3). It is shown that absence of P

leads to abolition of G1 arrest or apoptosis in response to ionizing radiation and DNA damaging agents (4).

Some reports have associated loss of P53, with in- creased sensitivity to chemotherapy, whereas others to increased chemoresistance (4 – 6). Cervical cancer che- motherapy in vivo improved in cases with high P expression in the tumor tissue (7, 8). It has been shown that transactivation and DNA-binding affinity of acti- vator protein (AP-1) as well as P53 can be modulated by both posttranslation modifications as well as by alteration of intracellular redox state (9, 10). Cervical cancer is associated with low antioxidant status (11). Vitamin C has been suggested to play a protective role in development of cervical intraepithelial neoplasia (CIN) (12), however, its role in the treatment of cervical cancer has not been studied. To investigate the effect of vitamin C on the tran- scriptional regulation of HPV E6/E7 oncogene expres- sion, HPV-18 positive Hela cells were treated with noncytotoxic amounts of vitamin C. The findings sug- gest that vitamin C caused downregulation of the viral oncoprotein E6, which was paralleled by a decrease of AP-1 members c-jun and c-fos in a dose- and time- dependent manner. The downregulation of E6 resulted in the up regulation of proapoptotic, P53 and Bax pro- teins but downregulation of apoptotic inhibitor Bcl-2. There was also significant decrease in telomerase ac- tivity. Vitamin C also increased the susceptibility/ apoptosis induced by cisplatin and etoposide.

MATERIALS AND METHODS

Cell culture. The human cervical carcinoma cell line (Hela) was obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10% fetal calf serum and antibiotics in a humidified atmosphere of 5% CO 2 in air at 37°C. Logarithmically growing cells were used for all experiments. Drug treatment and cell viability assay. 1 3 10 4 cells seeded in 96-well microtiter plates were treated with in vivo achievable con- centrations of vitamin C ranging from 0.1 mM to 10 mM and/or chemotherapeutic drugs i.e., cisplatin (2–100 mM), etoposide (2– mM), adriamycin (0.01–10 mM), bleomycin (0.0004 – 0.4 U/ml). The growth inhibitory effect of vitamin C and chemotherapeutic drugs was assessed by the MTT assay. After 48 h of incubation, 100 ml of 5 mg/ml of MTT was added followed by incubation for 4 h at 37°C. The

(^1) To whom correspondence should be addressed at Department of

Biochemistry, Room No 3027-A, All India Institute of Medical Sci- ences, Ansari Nagar, New Delhi-110029, India. Fax: 91-11-6862663. E-mail: singh_neeta@hotmail.com.

Biochemical and Biophysical Research Communications 282, 409 – 415 (2001)

doi:10.1006/bbrc.2001.4593, available online at http://www.idealibrary.com on

409 0006-291X/01 $35.

Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

formazan crystals thus formed were dissolved in DMSO and the absorbance measured at 570 nm with an ELISA reader. Immunocytochemistry. HeLa cells were grown on glass cover slips and treated with vitamin C (0.1 mM–10 mM). The cells were washed in PBS, fixed in ice-cold acetone, and methanol (1:1) for 10 min, and incubated for 1 h with primary antibodies c-Jun, c-Fos, P53, HPV-18-E6, Bax, and Bcl2 (Santa Cruz Biotechnology, USA). Sub- sequently the respective secondary antibody conjugated with alka- line phosphatase (AP) was added, the colour developed using BCIP and NBT (Promega, USA), and visualized under a light microscope (200 3 magnification). Western blot analysis. Equal amounts of protein extract was elec- trophoresed on 12–15% SDS–PAGE gels and electrotransferred to nitrocellulose membrane. Western blotting was done using the kit from Promega, USA, as described by the manufacturer. The blots were scanned by UMAX Gemini D-16 scanner and comparative den- sitometric analysis was done using NIH 1.52 image analysis soft- ware. Detection of apoptosis. Apoptotic cells were visualized by TUNEL assay using the DeadEnd colorimetric detection system (Promega, USA) and by flow cytometry as described earlier (13). Flow cytometry was performed using an EPICS XL-MCL flow cytometer (Coulter Electronics, Miami, FL) and the data was analyzed using WinMDI 2.8 software. Assay of telomerase activity. This was measured using PCR- based ELISA kit (Roche Molecular Biochemicals, Germany). The samples were lysed, and an aliquot containing 2 mg of protein was used for the assay. Telomerase positive embryonic kidney cell line (293) and untreated Hela cells were used as positive controls while heat inactivated Hela cell extract was used as negative control. The activity detected was expressed in relation to the positive control Hela cells.

RESULTS

Vitamin C Downregulates E6 Oncoprotein

To study the effect of vitamin C on transcriptional activity of HPV, HeLa cells were treated with vitamin

C doses ranging from 0.1 mM to 10 mM for 3 to 48 h. A dose of 1 mM vitamin C downregulated E6 without being toxic, as observed by immunocytochemistry and Western blot analysis (Figs. 1 and 2), and hence this dose was used for all further experiments. Immunocy- tochemistry revealed nuclear and cytoplasmic expres- sion of P53 (Fig. 1). b-Actin levels in all the treatments were found to be normal.

Vitamin C Downregulates c-Jun/c-Fos

Since HPV is under the transcriptional control of AP-1, we looked at the protein expression of c-jun/c-fos AP-1 heterodimer, a predominant form of AP-1 com- plex in Hela cells. Vitamin C treatment decreased the expression of both c-fos and c-jun, but more signifi- cantly that of c-fos (Fig. 2), starting at 3– 6 h and reaching the lowest level of 8.96% for c-fos and 47.3% for c-jun at 36 h, but recovered at 48 h.

Vitamin C Upregulates P53, Bax, and Downregulates Bcl

Since bax and bcl2 are the down stream targets of P53, we looked at their protein expression. The ob- served upregulation of P53 was followed by increase in Bax protein expression which reached a maximum of 328% at 24 h as compared to the control and this was sustained till 36 h. Concurrently, the expression of Bcl2 showed a 50% decrease after an initial increase at 3– 6 h (Fig. 2).

Vitamin C Produces Partial G1/S Block

The dose of vitamin C (1 mM) which was able to upregulate P53, induced a partial G1/S block, a 14%

FIG. 1. E6 and P53 protein expression as detected by immuno- cytochemistry (a) E6 expression in untreated control Hela cells shows predominance of perinuclear and cytoplasmic expression (b) E6 expression 24 h after treatment with 1 mM vitamin C (c) P expression in untreated control Hela cells (d) P53 expression 24 h after 1 mM vitamin C treatment shows both nuclear and cytoplasmic expression, nuclear expression is more in cells undergoing apoptosis.

FIG. 2. Western blot analysis of E6, c-Fos, c-Jun, P53, Bax, and Bcl2 protein expression in Hela cells after treatment with 1 mM vitamin C for various time periods, and their percentage expression compared to the control taken as 100%.

5B). There was no increase in G1/S arrest, however, partial S/G2 block was seen on vitamin C priming for 12 h. Interestingly, vitamin C priming for 36 h po- tentiated the G2/M arrest produced by cisplatin and this was accompanied by maximum apoptosis. This indicates that the upregulated P53 is not sufficient to produce a G1/S arrest, but is sufficient to trigger increased apoptosis. Priming with high dose vitamin C (1 mM) produced increased cell death when combined with low dose of cisplatin (2–10 mM), but cell death was decreased on increasing the dose of cisplatin (25–100 mM) (Fig. 4B). The percentage apoptosis decreased from 74.4 to 56% after simultaneous administration of 50 mM cisplatin with 1 mM vitamin C, and decreased from 97 to 73.6% when cisplatin (50 mM) was administered after prim- ing with vitamin C (1 mM) for 24 h (Fig. 4B). This indicates that the dose of vitamin C and cisplatin as well as the duration of priming was critical for the additive effect. The percentage apoptosis increased from 43.1% with 2 mM etoposide to 66% after priming with 1 mM vitamin C for 36 h (Fig. 4c). Similarly priming with 1 mM vitamin C for 36 h increased the percentage apoptosis by 16% with 0.1 mM adriamycin and by 18% with .0004 U/ml of bleomycin (data not shown). Thus the nature of cell-cycle arrest and the induction of apoptosis appears to be dependent on the type of chemotherapeutic drug.

Vitamin C Treatment Decreases Telomerase Activity

Reactivation of telomerase is a significant step in tumorigenesis and downregulation of telomerase activ- ity is a new approach of cancer control. Perhaps this is the first demonstration showing a significant decrease

in telomerase activity following vitamin C (1 mM) treat- ment, which was detectable as early as 6 h and reached 50% of control by 36 h (Fig. 6). The decrease in telo- merase activity coincided with the decrease in E6 and increase in P53 protein expression. This indicates that restoration of normal cell-cycle control mechanism by decreasing HPV transcription can decrease the telo- merase activity.

DISCUSSION

The cause for poor responsiveness to chemotherapy lies in the etiopathogenesis of cervical cancer i.e., HPV infection and loss of tumor suppressor gene function due to inactivation of P53 and Rb by HPV encoded viral oncoproteins. The restoration of P53 levels could be a potential strategy to increase chemoresponsiveness. However, there are conflicting reports regarding the role of P53 and chemosensitivity (4 – 6). Different au- thors have adopted different strategies to decrease E (14, 15) and stabilize P53 levels. We selected vitamin C based on the fact that it prevents the development of CIN to cervical cancer and decreases methylcholan- threne (MCA) induced cervical cancer in mice (12, 16). Vitamin C at low doses was seen to significantly de- crease c-Fos/c-Jun AP-1 dimer followed by decrease in E6 and increase in p53 protein. The level of Fra-1 was not estimated since it expression is low in Hela cells. We found that the increase in P53 resulted in upregu- lation of pro-apoptotic Bax and downregulation of anti apoptotic Bcl2, but was not sufficient to induce apop- tosis. Cisplatin, the single most active drug against cervi- cal cancer, was found to produce maximum additive affect in vitro on vitamin C pretreatment, followed by etoposide, whereas adriamycin and bleomycin showed only moderate effects. The probable reason for this could be that adriamycin and bleomycin themselves produce reactive oxygen species and are known to show chemoresistance in the presence of increased antioxi- dant status of the cell (17). Another important finding of our study was that the combination of high doses of vitamin C and cisplatin could decrease the effect of chemotherapy. The reason for this is not known al- though it is known that vitamin C at low doses acts as antioxidant and at high doses as a pro-oxidant (18). However, this finding is in congruence with a report, which states that hydrogen peroxide lowers the degree of cell killing by cisplatin (19). Our findings suggest that priming with low dose of vitamin C can have a significant additive effect particularly with low dose of in vivo achievable chemotherapeutic drugs, as shown by increased apoptosis. Although vitamin C showed increase in the levels of P53, it was unable to produce a very significant G1/S arrest. There was also no G1/S arrest in re- sponse to chemotherapeutic drugs indicating that

TABLE 2

Cellular Viability after Cisplatin Treatment Following

Priming with 1 mM Vitamin C

Vitamin C dose/ priming duration

CP

0 mM

CP

2 mM

CP

10 mM

CP

25 mM

CP

100 mM

0 mM 100 88.5 58.3 38.3 29. 1 mM/0 h 116 83.16 50.48 36.68 30. 1 mM/6 h 156.7 90.2 57.2 31.8 30. 1 mM/12 h 97.7 54.4 46.48 26.4 21. 1 mM/24 h 93.7 *39.6 *36.5 31.7 21. 1 mM/36 h 90.7 *37.4 *35.3 30.1 19.

Note. Viability of Hela cells after priming with 1 mM vitamin C for 6 –36 h followed by cisplatin treatment with various doses for 48 h. The percentage viability is expressed in comparison to untreated controls. The data is the mean of 3 separate experiments.

  • These values are significantly different from the control as seen by Student’s t -test ( P , 0.01).

FIG. 4. (A) Effect of 1 mM vitamin C priming on cisplatin induced apoptosis (a) Control: 0 – 48 h (b) Vitamin C 1 mM: 0 – 48 h (c) Cisplatin 2 mM: 0 – 48 h (d) Vitamin C 1 mM: 0 –12 h 1 Cisplatin 2 mM: 12– 60 h (e) Vitamin C 1 mM: 0 –24 h 1 Cisplatin 2 mM: 24 – h (f) Vitamin C 1 mM: 0 –36 h 1 Cisplatin 2 mM: 36 – 84 h. (B) Effect of high dose of vitamin C (1 mM) on cisplatin induced apoptosis (a) Cisplatin 50 mM: 0 – 48 h (b) Vitamin C 1 mM 1 Cisplatin 50 mM: 0 – 48 h (c) Cisplatin 50 mM: 24 –72 h (d) Vitamin C 1 mM: 0 – h 1 Cisplatin 50 mM: 24 –72 h. (C) Effect of 1 mM vitamin C priming on etoposide induced apoptosis (a) etoposide 2 mM: 0 – 48 h (b) vitamin C 1 mM: 0 –12 h 1 etoposide 2 mM: 12– 60 h (c) vitamin C 1 mM: 0 –24 h 1 etoposide 2 mM: 24 –72 h (d) vitamin C 1 mM: 0 – h 1 etoposide 2 mM: 36 – 84 h.

num complexes in a human ovarian cancer cell line. Mol. Phar- macol. 57, 503–511.

  1. Garzetti, G. G., Ciavattini, A., Provinciali, M., Di Stefano, G., Lucarini, G., Goteri, G., and Biagini, G. (1996) Expression of P and apoptosis of tumor cells in locally advanced cervical carci- noma after Cisplatin based neoadjuvant chemotherapy. Antican- cer Res. 16, 3229 –3234.
  2. Miyake, H., Hara, I., Hara, S., Arakawa, S., and Kamidono, S. (2000) Synergistic chemosensitization and inhibition of tumor growth and metastasis by adenovirus-mediated P53 gene trans- fer in human bladder cancer model. Urology 56, 332–336.
  3. Gomez Del Arco, P., Martinez-Martinez, S., Calvo, V., Armesilla, A. L., and Redondo, J. M. (1997) Antioxidants and AP-1 activa- tion: A brief overview. Immunobiology 198, 273–278.
  4. Liu, M., Pelling, J. C., Ju, J., Chu, E., and Brash, D. E. (1998) Antioxidant action via P53-mediated apoptosis. Cancer Res. 58, 1723–1729.
  5. Ho, G. Y., Palan, P. R., Basu, J., Romney, S. L., Kadish, A. S., Mikhail, M., Wassertheil-Smoller, S., Runowicz, C., and Burk, R. D. (1998) Viral characteristics of human papillomavirus in- fection and antioxidant levels as risk factors for cervical dyspla- sia. Int. J. Cancer 78, 594 –599.
  6. Kwasniewska, A., Tukendorf, A., and Semczuk, M. (1996) Fre- quency of HPV infection and the level of ascorbic acid in serum of women with cervix dysplasia. Med. Dosw. Mikrobiol. 48, 183–
  7. Khanna, N., Reddy, V. G., Tuteja, N., and Singh, N. (2000) Differential gene expression in apoptosis: Identification of ribo- somal protein S29 as an apoptotic inducer. Biochem. Biophys. Res. Commun. 277, 476 – 486.
  8. Maehama, T., Patzelt, A., Lengert, M., Hutter, K. J., Kanazawa, K., Hausen, H., and Rosl, F. (1998) Selective down-regulation of human papillomavirus transcription by 2-deoxyglucose. Int. J. Cancer 76, 639 – 646.
  9. Narayanan, B. A., Holladay, E. B., Nixon, D. W., and Mauro, C. T. (1998) The effect of all-trans and 9-cis retinoic acid on the steady state level of HPV16 E6/E7 mRNA and cell cycle in cervical carcinoma Cells. Life. Sci. 63, 565–573.
  10. Das, P., Rao, A. R., and Srivastava, P. N. (1993) Influence of ascorbic acid on MCA-induced carcinogenesis in the uterine cer- vix of mice. Cancer Lett. 72, 121–125.
  11. Kumar, D., Kirshenbaum, L., Li, T., Danelisen, I., and Singal, P. (1999) Apoptosis in isolated adult cardiomyocytes exposed to adriamycin. Ann. NY Acad. Sci. 874, 156 –168.
  12. Sestili, P., Brandi, G., Brambilla, L., Cattabeni, F., and Cantoni, O. (1996) Hydrogen peroxide mediates the killing of U937 tumor cells elicited by pharmacologically attainable concentrations of ascorbic acid: Cell death prevention by extracellular catalase or catalase from cocultured erythrocytes or fibroblasts. J. Pharma- col. Exp. Ther. 277, 1719 –1725.
  13. Shacter, E., Williams, J. A., Hinson, R. M., Senturker, S., and Lee, Y. J. (2000) Oxidative stress interferes with cancer chemo- therapy: Inhibition of lymphoma cell apoptosis and phagocytosis. Blood 96, 307–313.
  14. Hagopian, G. S., Mills, G. B., Khokhar, A. R., Bast, R. C., Jr., and Siddik, Z. H. (1999) Expression of P53 in cisplatin-resistant ovarian cancer cell lines: Modulation with the novel platinum analogue (1R, 2R-diaminocyclohexane)(trans-diacetato)(dichloro)- platinum (IV). Clin. Cancer Res. 5, 655–663.
  15. Choo, C. K., Ling, M. T., Chan, K. W., Tsao, S. W., Zheng, Z., Zhang, D., Chan, L. C., and Wong, Y. C. (1999) Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames. Prostate 40, 150 –158.
  16. Kanaya, T., Kyo, S., Hamada, K., Takajura, M., Kitagawa, Y., Harada, H., and Inoue, M. (2000) Adenoviral expression of P represses telomerase activity through down regulation of human telomerase reverse transcriptase transcription. Clin. Cancer Res. 6, 1239 –1247.