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Vol. 2, 165-171, February 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

p53-independent Response to Cisplatin and Oxaliplatin in MMTV-ras Mouse Salivary Tumors1

Thierry Petit2, David J. Bearss3, Dean A. Troyer, Ruben M. Munoz3 and Jolene J. Windle4

Cancer Therapy and Research Center [T. P., R. M. M., J. J. W.], Departments of Cellular and Structural Biology [D. J. B., J. J. W.] and Pathology [D. A. T.], The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229

4 To whom requests for reprints should be addressed, at Massey Cancer Center, Virginia Commonwealth University, P.O. Box 980037, Richmond, VA 23298-0037. Phone: (804) 828-5843; Fax: (804) 828-5836; E-mail: jjwindle{at}hsc.vcu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A transgenic mouse tumor model was used to investigate the role of p53 in tumor response to two different platinum-based chemotherapeutic agents: (a) cisplatin and (b) oxaliplatin, a diaminocyclohexane platine recently introduced into the clinic. MMTV-v-Ha-ras transgenic mice were interbred to p53-deficient mice to generate mice that develop salivary tumors either possessing or lacking p53. Tumor-bearing mice were then treated on either a 9-day schedule to assess overall tumor growth response or on a short-term treatment schedule to assess effects on cell cycle parameters and apoptosis. Both agents induced significant apoptosis and promoted overall tumor regression, regardless of the p53 status of the tumor. This is in contrast to previous studies using this model in which treatment with paclitaxel or doxorubicin promoted tumor growth arrest but not apoptosis. These findings indicate that even in the context of an activated ras gene that potentially mediates suppression of apoptosis, both cisplatin and oxaliplatin are capable of promoting an efficient p53-independent tumor response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The p53 tumor suppressor gene is a critical determinant of many tumor properties, including response to a variety of treatment modalities. Mutations of the p53 gene are present in ~50% of all human tumors (1) and correlate with several markers of poor prognosis, including high S phase fraction, aneuploidy, high histological grade, and metastasis (2). Functional p53 can promote either an apoptotic or growth arrest response after DNA damage, and both of these responses are impaired in cells lacking wild-type p53 function (3). Accordingly, p53-deficient tumor cells display decreased sensitivity to many chemotherapeutic agents, most notably those that function by inducing DNA damage (2, 4–6). It is therefore of considerable clinical importance to determine the extent to which any given chemotherapeutic agent is dependent on p53 status for its antitumor activity.

Cisplatin [cis-diamminedichloroplatinum(II)] is a member of a family of platinum-based compounds that form various intra and interstrand adducts with DNA (7). It has a broad spectrum of antitumor activity and is widely used clinically in the treatment of many types of cancer (8). However, its use is limited by the fact that many tumors are either inherently resistant or acquire resistance on recurrence after an initial response. Based on its mechanism of action, cisplatin might be expected to function in a p53-dependent manner. Consistent with this prediction, sensitivity to cisplatin was shown to correlate with the presence of wild-type p53 function in a NCI5 panel of 60 human tumor cell lines, although there were multiple exceptions (6). In addition, p53 inactivation has been shown to promote increased resistance to cisplatin in a variety of tumor cell types (4, 9–14). Other studies, however, have shown no correlation between p53 status and response to cisplatin (15, 16) or have actually demonstrated the opposite correlation, that p53 mutation promotes increased sensitivity to cisplatin (17–20). Furthermore, one study demonstrated that in SaOS-2 osteosarcoma cells, p53 function was associated with increased cisplatin sensitivity under high serum growth conditions but with decreased sensitivity under low serum conditions (21). Thus, the relationship between p53 status and cisplatin cytotoxicity appears to be complex and may depend on several factors, including tumor cell type, activation of specific signaling pathways, or the presence of other genetic alterations.

We have used previously a transgenic/knockout mouse tumor model to investigate the role of p53 in response to chemotherapeutic agents (22, 23). MMTV-v-Ha-ras transgenic mice (24) were interbred to p53 knockout mice (25) to generate mice that develop mammary and salivary tumors either possessing or lacking p53 function. In the present study, we have used this in vivo model system to evaluate the role of p53 status on tumor responsiveness to cisplatin, as well as to a clinically active DACH platinum compound, oxaliplatin (26). Oxaliplatin was chosen because it was shown to have a markedly different spectrum of activity than cisplatin using the Drug Discovery program from the NCI (27). In this model system, we find that tumor response to both agents, although subtly different, is largely p53 independent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice.
MMTV-v-Ha-ras mice were obtained from Charles River Laboratories, and p53-/- mice were obtained from Taconic Laboratories. Although these mice were originally obtained in inbred genetic backgrounds (FVB and C57BL/6, respectively), they were subsequently maintained in our laboratory in a mixed genetic background containing C57BL/6, BALB/c, and FVB contributions. Offspring from matings between MMTV-v-Ha-ras and p53-/- or p53+/- mice were screened using PCR to determine their ras transgene and p53 gene status, as described previously (28). Consistent with data published previously, MMTV-ras/p53+/+ mice developed mammary and salivary adenocarcinomas with a median age of onset of ~8 months, whereas MMTV-ras/p53-/- mice developed primarily salivary adenocarcinomas with a median age of onset of ~2.5 months (28). Therefore, only salivary tumors were used in this study to permit a direct comparison between the same tumor type arising in mice of the two genotypes.

Drug Treatment of Tumor-bearing Mice.
Animals were monitored visually twice weekly for the presence of tumors. Once tumors were detected, tumor growth was monitored by taking caliper measurements in two perpendicular dimensions. Tumor volume (mm3) was calculated according to the formula (W2 x L)/2, where W (width) and L (length) are in millimeters and L >= W. Animals were placed on study when one or more tumors reached ~200–800 mm3. Cisplatin and oxaliplatin were freshly prepared each week. For tumor growth studies, cisplatin (Bristol-Myers Oncology Division, Princeton, NJ) and oxaliplatin (Sanofi-Synthelabo Research, Great Valley, PA) were administered i.p. at 2.5 and 5 mg/kg, respectively, qd for 9 days, and tumor growth was monitored by daily caliper measurements. These doses are below the maximum tolerated dose for this treatment schedule and resulted in minimal morbidity or mortality. Tumor MGRs for untreated tumors were calculated according to the formula [(sum area under the curve) - (vol1 x (dayn - day1)]/(dayn - day1)2, where the area under the curve was calculated according to the formula [(vol1 + vol2)/2] x (day2 - day1). Tumor responses to cisplatin and oxaliplatin were determined by calculating the time required for each tumor to regress to half of its original size, and the values were compared across groups using the Student t test.

For the measurement of apoptosis and cell cycle parameters, a single i.p. injection of cisplatin at 7.5 mg/kg or oxaliplatin at 30 mg/kg was administered, and the animals were sacrificed 48 h later. These doses are near but below the maximum tolerated dose for a single treatment.

Flow Cytometry.
On sacrificing a tumor-bearing mouse, a 25–50-mg piece of tumor tissue was frozen in liquid nitrogen and stored at -80°C until the time of analysis. Tissue was processed and stained with propidium iodide using a modified Krishan technique (29) and analyzed with an EPICS ELITE flow cytometer (Coulter Cytometry, Miami, FL) as described previously (28). Histograms were analyzed for cell cycle compartments using MultiCycle-PLUS Version 3.0 (Phoenix Flow Systems, San Diego, CA). A minimum of 50K events was collected to maximize statistical validity of the compartmental analysis. Differences in S and G2-M phase fractions between groups were evaluated using the Student t test.

Apoptosis Analysis.
Tumor samples were collected and fixed in 10% buffered formalin (pH 7). The samples were then processed and embedded in paraffin. The TUNEL method was used to label apoptotic cells in situ (30), and sections were evaluated by light microscopy. Positively labeled cells within a 10 x 10 mm grid in the eyepiece were counted in 10 x450 fields. Areas of the tumor that appeared necrotic by light microscopy were excluded. The apoptotic cells in non-necrotic regions generally appeared to be distributed uniformly throughout viable regions of the tumor. The total number of tumor cells in each field ranged from 500 to 800, depending on the histological pattern of the tumor. The percentage of apoptosis was calculated based on an average of 670 cells/field. Differences in apoptosis values between groups were evaluated using the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Salivary Tumor Response to Long-Term Treatment with Cisplatin and Oxaliplatin.
As we have reported previously (28), tumors arising in MMTV-ras/p53-/- mice have an MGR nearly double that of tumors arising in MMTV-ras/p53+/+ mice. In untreated mice, the tumor MGR was 51.6 ± 13.5 mm3/day for ras/p53+/+ tumors and 87 ± 16.2 mm3/day for ras/p53-/- tumors (Fig. 1, A and D). Salivary tumors arising in both MMTV-ras/p53+/+ and MMTV-ras/p53-/- mice regressed in response to 9 days of cisplatin treatment (Fig. 1, B and E). The mean time required for tumors to regress to half of their starting volume was 7.8 ± 0.6 days for ras/p53+/+ tumors and 8.3 ± 0.5 days for ras/p53-/- tumors. Similarly, tumors of both genotypes responded efficiently to oxaliplatin (Fig. 1, C and F), with a mean time for regression to half of the starting volume of 7.2 ± 0.6 days for ras/p53+/+ tumors and 7.5 ± 0.3 days for ras/p53-/- tumors. There was no significant difference between ras/p53+/+ and ras/p53-/- tumors in their response to either agent, indicating that these agents function in a largely p53-independent manner in this tumor model. Interestingly, cisplatin treatment of tumors of either genotype consistently resulted in a lag of 2–3 days before tumors began to regress, and during this time, tumors either plateaued or continued to grow. In contrast, treatment with oxaliplatin resulted in tumor regression within the 1st day of treatment. However, when measured as the time to regress to half the starting volume, there was no significant difference for tumors of either genotype in their response to cisplatin versus oxaliplatin.



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  		Fig. 1. Salivary tumor growth response to cisplatin and oxaliplatin in MMTV-ras/p53+/+ and MMTV-ras/p53-/- mice. Tumor-bearing mice were treated for nine consecutive days (starting on day 1) with cisplatin or oxaliplatin, and tumor growth was monitored by daily caliper measurements. Each line represents the growth of an individual tumor. The numbers of mice in each treatment group were: 9 untreated MMTV-ras/p53+/+ mice, 10 untreated MMTV-ras/p53-/- mice, 9 MMTV-ras/p53+/+ mice treated with cisplatin, 7 MMTV-ras/p53-/- mice treated with cisplatin, 9 MMTV-ras/p53+/+ mice treated with oxaliplatin, and 5 MMTV-ras/p53-/- mice treated with oxaliplatin.

 
Cell Cycle Changes in Tumor Response to Cisplatin and Oxaliplatin.
As we reported previously (28), tumors from untreated MMTV-ras/p53-/- mice displayed increased S and G2-M phase fractions and a corresponding decrease in the proportion of cells in G1, compared with untreated ras/p53+/+ tumors (Fig. 2), consistent with the involvement of p53 in a G1 to S phase checkpoint. Treatment of ras/p53+/+ tumors with either cisplatin or oxaliplatin resulted in highly significant increases in the percentage of cells in both S and G2-M phase (P = 0.03 for cisplatin-induced increase in S phase; P = 0.0001 for oxaliplatin), rather than an accumulation of cells in G1, indicating that these agents failed to activate the p53-dependent G1-S checkpoint (Fig. 2A). A similar although less dramatic shift in cell cycle distribution was observed in ras/p53-/- tumors treated with either cisplatin or oxaliplatin as compared with untreated ras/p53-/- tumors (Fig. 2B). However, in the ras/p53-/- tumors, only the increases in G2-M phase fractions were statistically significant (P = 0.02 for cisplatin-induced increase in G2-M; P = 0.0008 for oxaliplatin). The accumulation of cells in S and G2-M is consistent with studies demonstrating that cisplatin-induced DNA damage slows the progression of cells through S phase and eventually promotes a G2-M arrest (19, 31).



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  		Fig. 2. The effects of cisplatin and oxaliplatin on cell cycle profiles of ras/p53+/+ (A) and ras/p53-/- (B) salivary tumors. The relative fractions of cells in G1, S, and G2-M phases of the cell cycle were determined using flow cytometric analysis of propidium iodide-stained tumor cells from untreated mice or mice sacrificed 48 h after a single treatment of cisplatin or oxaliplatin. The actual cell cycle fractions (G1, S, and G2-M) ± the SE for each of the treatment groups were: 94.2 ± 0.9, 4.3 ± 0.7, and 1.5 ± 0.3 (ras/p53+/+, untreated; n = 13 tumors); 81.6 ± 4.3, 12.4 ± 4.4, and 6 ± 1.5 (ras/p53+/+, cisplatin; n = 8 tumors); 81.1 ± 3.2, 14.4 ± 3.2, and 4.6 ± 0.8 (ras/p53+/+, oxaliplatin; n = 10 tumors); 84.2 ± 1.7, 10.9 ± 1.5, and 4.9 ± 0.7 (ras/p53-/-, untreated; n = 16 tumors); 78.8 ± 2.5, 12.2 ± 1.5, and 8.8 ± 1.3 (ras/p53-/-, cisplatin; n = 21 tumors); and 74.7 ± 2.4, 13.6 ± 1.6, and 11.7 ± 1.8 (ras/p53-/-, oxaliplatin; n = 19 tumors).

 
Induction of Tumor Cell Apoptosis in Response to Cisplatin and Oxaliplatin.
The levels of spontaneous apoptosis in salivary tumors from untreated MMTV-ras mice were uniformly low, regardless of p53 status (Fig. 3). In addition, we have shown previously that these tumors are resistant to the induction of apoptosis by either doxorubicin or paclitaxel (22), agents that are potent inducers of apoptosis in other systems (32–34). In contrast, both cisplatin and oxaliplatin promoted a significant induction of apoptosis in ras/p53+/+ tumors compared with untreated tumors (3.8-fold for cisplatin; 6.5-fold for oxaliplatin). Interestingly, oxaliplatin induced a higher level of apoptosis than cisplatin in these tumors (P = 0.05). Furthermore, this apoptosis was largely p53 independent, because both agents also induced apoptosis in ras/p53-/- tumors (Fig. 3). The extent of induction by cisplatin was identical to that seen in ras/p53+/+ tumors, and although there appeared to be a decreased induction of apoptosis by oxaliplatin in ras/p53-/- as compared with ras/p53+/+ tumors, this difference was not significant.



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  		Fig. 3. Apoptotic response of ras/p53+/+ and ras/p53-/- salivary tumors to cisplatin and oxaliplatin. TUNEL analysis was used to determine the percentage of cells undergoing apoptosis in untreated tumors and tumors 48 h after a single treatment with cisplatin or oxaliplatin. TUNEL-positive cells were identified by light microscopy and quantitated as described in "Materials and Methods." Error bars represent the SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objective of the present study was to investigate the potential association between p53 status and tumor response to two different platinum compounds, cisplatin and oxaliplatin, in an in vivo tumor model system, because cell culture studies have produced conflicting results with respect to the role of p53 in tumor response to cisplatin (4, 6, 9–21). We used a transgenic/knockout mouse salivary tumor model in which MMTV-v-Ha-ras transgenic mice (24) were interbred to mice in which the p53 gene has been inactivated (25) to produce ras-driven salivary tumors that either possess or lack p53 (28). These mice represent a genetically accurate model for human salivary tumors, in which both ras and p53 are found frequently to be mutated and correlate with higher tumor grade (35–40). Interestingly, although Ki-ras is the ras gene most frequently mutated in human tumors overall (41, 42), the Ha-ras gene is most often found to be mutated in human salivary tumors (35, 43). The salivary tumors arising in these mice also represent a histologically accurate model for a subset of human salivary tumors (44). Salivary tumors are one of the most morphologically complex human tumor types, involving multiple primary sites and histological subtypes (45), and the salivary tumors arising in the MMTV-v-Ha-ras mice closely resemble human acinic cell carcinomas (44). The initial treatment for salivary tumors of all subtypes is almost always surgical and is in some cases followed by radiation therapy. However, in cases involving distant metastases, chemotherapy is included and generally involves the use of combination regimens that include cisplatin (45).

In this mouse model system, both cisplatin and oxaliplatin exhibited equal antitumor activity in ras/p53+/+ and ras/p53-/- salivary tumors, indicating that the tumor growth response was largely p53 independent. It was thus of interest to determine whether individual mechanistic components of the response, cell cycle arrest and/or apoptosis, were similarly p53 independent. p53 plays an important role in mediating a G1-S cell cycle arrest after DNA damage (3). Using this same tumor model system in a previous study, it was demonstrated that the DNA-damaging agent doxorubicin induced an accumulation of cells in G1 in ras/p53+/+ but not ras/p53-/- tumors, reflecting a loss of the p53-dependent G1-S checkpoint in the p53-deficient tumors (22). Rather, the ras/p53-/- tumors underwent an apparent p53-independent G2 block in response to this DNA-damaging agent. These results are consistent with a large body of data indicating that doxorubicin activates p53-dependent cell cycle checkpoints (46). In the same study, treatment of MMTV-ras tumors with the agent paclitaxel resulted in an accumulation of cells in G2-M, regardless of p53 status, consistent with the reported p53-independent activity of paclitaxel (6). These data indicate that the MMTV-ras tumor model can predict whether an agent functions in a p53-dependent or -independent manner. Thus, if cisplatin- or oxaliplatin-induced DNA damage promoted a p53-dependent response, one would expect to see an accumulation of cells in G1 in ras/p53+/+ tumors but not in ras/p53-/- tumors. Instead, treatment with either cisplatin or oxaliplatin resulted in a decreased percentage of cells in G1 and an increased percentage of cells in S and G2-M, regardless of p53 status. This indicates that the cell cycle response to cisplatin and oxaliplatin is largely p53 independent in MMTV-ras salivary tumors. The fact that these compounds promote both tumor regression and an increase in S and G2-M phase fractions may seem paradoxical. However, it is likely that this observation reflects retarded progression through S phase, rather than increased proliferation, because it has been reported previously that cisplatin-induced DNA damage slows the progression of cells through S phase and eventually promotes a G2-M arrest (19, 31).

The apoptotic response of tumor cells to chemotherapeutic treatment has been directly correlated to overall response with respect to tumor growth inhibition or regression (47, 48). This suggests that apoptotic cell death represents a major component of the cytotoxicity induced by chemotherapeutic agents and that genetic alterations conferring resistance to apoptosis may contribute to multidrug resistance (49, 50). A central function of p53 is its ability to mediate cell death by apoptosis (3), and p53-dependent apoptosis has been suggested to be a major effector of response to chemotherapy (51). In ras/p53+/+ salivary tumors, treatment with either cisplatin or oxaliplatin induced a statistically significant increase in the levels of apoptosis as compared with untreated tumors. However, a similar induction of apoptosis was also observed in ras/p53-/- tumors. Thus, neither the overall tumor growth response nor the cell cycle and apoptotic responses to cisplatin and oxaliplatin seen in this model are p53 dependent.

The ras oncogenes have been shown to increase resistance to apoptosis in many contexts (52–56), and an increasing body of data suggests that signaling from Ras is an important factor in the response of cells to DNA-damaging agents, including platinum compounds (57). Thus, it might be expected that tumors possessing activated ras genes would exhibit an overall poorer response to chemotherapy. In fact, it is interesting to note that several of the tumor types associated with a high frequency of ras mutation, including pancreatic, lung, and colon cancers, generally exhibit a relatively chemoresistant phenotype (58). Consistent with ras-mediated suppression of apoptosis, the levels of spontaneous apoptosis in salivary tumors from MMTV-v-Ha-ras mice were uniformly low, regardless of p53 status (28). Furthermore, apoptosis was not significantly elevated in response to doxorubicin or paclitaxel in either ras/p53+/+or ras/p53-/- tumors (22). In fact, the only agent found previously to induce marked apoptosis in MMTV-ras tumors, independently of p53 status, was a Ras-inactivating farnesyltransferase inhibitor (23). Interestingly, in the present study, cisplatin and oxaliplatin treatment resulted in a significant induction of apoptosis, despite the presence of constitutively activated Ras. Furthermore, oxaliplatin induced a statistically significant higher level of apoptosis than cisplatin in ras/p53+/+ tumors.

Mechanistically, the antitumor activity of platinum compounds is generally regarded to result from their interaction with DNA, resulting in the formation of a variety of both intra and interstrand adducts (7). The major DNA adducts formed by cisplatin are intrastrand cross-links between neighboring bases, either between two guanines or a guanine and cytosine (59). Oxaliplatin results in the formation of similar DNA adducts, the main difference being the presence of the 1,2-DACH ring in the oxaliplatin adduct (60). This bulky DACH moiety that protrudes into the minor groove might induce poorly recognized lesions by DNA repair pathways (61). Multiple lines of evidence indicate that the cellular responses to these two agents may be different, leading to different profiles of activity, e.g., cisplatin and oxaliplatin have markedly different activity profiles when compared in the NCI’s 60 tumor cell line panel (27). Furthermore, in clinical trials, oxaliplatin has shown activity in a variety of cisplatin-resistant tumors (26). Differences in activity profiles between these two drugs may be explained in part by differences in their interaction with DNA damage repair pathways, because defects in the mismatch repair system are a major factor in cellular resistance to cisplatin (62) but do not appear to affect oxaliplatin activity (61). In the MMTV-ras salivary tumor model, cisplatin and oxaliplatin resulted in comparable levels of tumor regression and induced similar changes in cell cycle distribution and tumor cell apoptosis. However, there were interesting differences in the kinetics of tumor regression induced by these two agents. Although oxaliplatin induced an immediate regression, there was a delay of 2–3 days before cisplatin-treated tumors began to regress. These findings are consistent with the hypothesis that these two agents activate different cellular response pathways leading to cytotoxicity.

The present study indicates that in a ras-driven tumor model system, two different platinum-based chemotherapeutic agents induce a largely p53-independent tumor response. Given the considerable disparity in the literature regarding the requirement for p53 in tumor response to cisplatin, one must conclude that the relationship between p53 and response to platinum agents is much less straightforward than for many other DNA-damaging agents. Very likely, this relationship will be dependent on a number of other cellular factors, including alterations in DNA repair, growth, and apoptosis regulatory pathways.


   Acknowledgments
 
We thank Dr. Esteban Cvitkovic for his catalytic role in this study and thoughtful comments on this manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by American Cancer Society Grant DHP-150 (to J. J. W.) and a Medical Fellow Support Grant from Sanofi/ELF (to T. P.). Back

2 Present address: Centre Paul Strauss, 67085 Strasbourg, France. Back

3 Present address: Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721. Back

5 The abbreviations used are: NCI, National Cancer Institute; MMTV, mouse mammary tumor virus; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; DACH, diaminocyclohexane; MGR, mean growth rate. Back

Received 3/15/02; revised 12/ 2/02; accepted 12/ 5/02.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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