Cooperation of p53 loss of function and v-Ha-ras in transformation of mouse keratinocyte cell lines

Abstract

We previously demonstrated that after transduction with the v-Ha-ras oncogene and grafting onto nude mouse hosts, primary epidermal keratinocytes with a null mutation in the p53 gene form tumors with increased growth rates and predisposition to malignant conversion relative to p53 wild-type keratinocytes (Weinberg WC, et al., Cancer Res 54:5584-5592, 1994). To further explore the cooperation between p53 loss of function and activation of the ras oncogene, cell lines were established from the normal epidermises of newborn and adult p53-null mice, and parallel subclones were reconstituted with the p53val135 temperature-sensitive mutant. Reconstituted lines C, G, N, and V demonstrated functional p53 transcriptional activator activity at the wild-type-permissive temperature of 32 degrees C, compared with the hygromycin-selected control line X and parental p53-null lines NHK4 and AK1b. Hygromycin-selected subclones, but not the parental lines, made normal skin in vivo; all cell lines made carcinomas after introduction of v-Ha-ras, independent of p53 status. These cell lines were compared in vitro at 32 degrees C to maximize the amount of p53val135 in the wild-type conformation. Expression of v-Ha-ras did not consistently alter p53-mediated transcriptional activity, suggesting tat ras acts downstream or independently of p53. No correlation was observed between p53-mediated transcriptional activity and in vitro growth rates, colony formation after exposure to ultraviolet light, or suppression by normal neighboring keratinocytes. However, keratinocyte cell lines devoid of p53 and expressing v-Ha-ras formed colonies in soft agar; this was blocked at 32 degrees C in all cell lines reconstituted with p53val135. These keratinocyte lines provide a model for exploring the role of p53 and the interaction of p53 and ras in keratinocyte transformation.

p53 Is Necessary for the Apoptotic Response Mediated by a Transient Increase of Ras Activity

“…PKC, as a survivalpromoting factor, suppresses the activities of various proapoptotic factors, thereby helping cells to escape from p53 surveillance. The elimination of PKC activity releases the ap- Mutation of the p53 gene is a common event in tumorigenesis and often occurs in the late stage of malignancy (5,8,30,48). In various types of cells transformed by oncogenic ras, p53 appears to be mutated.…”

“…p53, as a transcriptional factor, also regulates the induction and function of several other genes, including Bax and p21, which participate in either p53-dependent apoptosis or growth arrest (28,36,37). It has been observed that mutations in both p53 and ras genes often occur in the same cancer cell (5,8,30,48). In colon cancer cells, Ras mutations have been demonstrated to precede p53 mutations (30).…”

p53 and Tumor Necrosis Factor α Regulate the Expression of a Mitochondrial Chloride Channel Protein

“…Section 1734 solely to indicate this fact. described previously (10), and these were maintained in medium containing 16% fibroblast conditioned Eagle's minimal essential medium with 8% chelexed serum and 0.05 mM Ca 2ϩ . Primary mouse keratinocytes and cell lines were treated with human recombinant TNF-␣ (R&D Systems, Minneapolis, MN) at final concentrations ranging from 10 to 100 ng/ml for the times detailed.…”

“…This implies that p53 is dispensable for normal skin development and differentiation. However, deletion of one or both p53 alleles enhances the establishment of immortal keratinocyte cell lines in vitro (10), suggesting that p53 could participate in keratinocyte mortality in a more subtle way, not involving expression of the major differentiation-related markers. To detect such a contribution of p53 to keratinocyte differentiation or mortality, differential expression of mRNA sequences was examined in keratinocytes isolated from p53 wild-type and null mice and induced to differentiate by Ca 2ϩ in vitro, a condition known to increase p53-dependent transcriptional activity.…”

The atypical E2F family member E2F7 couples the p53 and RB pathways during cellular senescence

“…p53 can also control the senescence of MEFs in a manner that directly restricts the ability of Ras to promote malignant transformation (Serrano et al 1997;Azzoli et al 1998). To examine the participation of E2F7 in this process, we developed multiple shRNAs targeting mouse E2f7 (Supplemental Fig.…”