A modified p53 overcomes mdm2-mediated oncogenic transformation: a potential cancer therapeutic agent

The antiproliferative activities of wild-type (wt) p53 are inhibited by mdm2 (murine double minute2) oncogene product. We tested growth suppression activity of p53 14/19, an engineered p53 variant, which does not bind mdm2 and is completely resistant to the inhibition by mdm2. p53 14/19, unlike wt p53, suppressed the growth of cancer cells that contain amplified mdm2 oncogene efficiently by direct DNA transfection or adenovirus-mediated gene transfer. In addition, p53 14/19 also inhibited the growth of several different cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We further examined the antioncogenic potencies of p53 14/19 in the rat embryo fibroblast cotransformation assay. Addition of wt p53 failed to cause any significant decrease in ras plus mdm2 foci counts. In contrast, cotransfection of p53 14/19 with ras and mdm2 significantly reduced foci number. In similar experiments, cotransfection of wt p53 or 14/19 p53 resulted in significant inhibition of oncogenic transformation in rat embryo fibroblast mediated by an activated ras plus c-myc, adenovirus E1A, or human papillomavirus E7 oncogenes. Therefore, these results suggest that p53 14/19 modified tumor suppressor gene may be a promising therapeutic agent for human cancers that express abnormally high levels of mdm2 oncogene product.     

Evidence for synergistic interactions between ras, myc and a mutant form of p53 in cellular transformation and tumor dissemination.

Mouse 10T1/2 cells were transfected with combinations of T24 H-ras, human c-myc and the proline 193 mutant form of p53. The three-gene ras/myc/p53 combination was significantly more efficient than single genes or double gene combinations in inducing transformed foci in vitro. An analysis of cell lines isolated after transfections with ras, ras/myc, ras/p53 and ras/myc/p53 indicated that the last combination contained significantly higher levels of ras protein than the other combinations, produced tumors in syngeneic mice with a shorter latency period, and exhibited an increased ability to form lung tumors in an in vivo experimental metastasis assay. Synergistic interactions between ras, myc and mutant p53 genes were observed in focus formation and metastasis assays, suggesting that the action of the three oncogenes in malignant transformation occurs along separate but interactive pathways. These results support a working model of oncogene cooperativity in which alterations in myc and p53 permit elevated expression of ras, which is important in a mechanism affecting both cellular transformation in vitro and tumor dissemination in vivo.     

Nuclear localization is essential for the activity of p53 protein.

p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function.     

High prevalence of HPV18 in correlation with ras gene mutations and clinicopathological parameters in cervical cancer studied from stained cytological smears.

The multi-event nature of carcinogenesis has led to extensive studies of oncogenes, onco-suppressor genes and viruses involved in human cancers. The collaboration of ras oncogene with HPV E6/E7 genes inducing full transformation of cervical keratinocytes has also been suggested. The purpose of this study was to detect the presence of codon 12 point mutations of ras genes, as well as to detect and identify the human papillomaviruses in stained smears of cervical malignancies and to correlate them with the clinicopathological parameters of the Greek patients. Specimens were obtained from 88 women, codon 12 point mutations of the K-ras (30%) and H-ras (10%) oncogenes, as well as HPV18 were detected at a higher rate than HPV16 (66% vs 7%) in cervical lesions by PCR-RFLP and PCR analysis, respectively. The statistical analysis of the data demonstrates correlation between the presence of K-ras mutations and FIGO stage and between FIGO stage and survival of the patients. It is suggested that ras activation combined with HPV infection may be an important step in the carcinogenesis of a substantial number of cervical carcinomas.     

Separation of immortalization and T24-ras oncogene cooperative functions of adenovirus E1a

An adenovirus 2 E1a gene coding for a protein of 243 (243R) amino acids can efficiently immortalize primary rat kidney (BRK) cells and cooperate with the activated cellular ras oncogene (T24 ras). A mutant (47-0) of the 243R gene that maps between amino acid residues 47-50 within a region that is highly conserved among the various adenovirus serotypes was found to be severely defective in immortalization. Despite the defect in immortalization, mutant 47-0 had the ability to cooperate with T24 ras in oncogenic transformation. These results suggest that the immortalization and the oncogene cooperation functions of the 243R are separable. Our results further suggest that the requirement for a separate immortalization function can be circumvented by oncogenic transformation and that the immortalization of cells transformed by E1a and T24 ras may be a secondary consequence of transformation by these two oncogenes.     

Inhibition of Ras oncogenic activity by Ras protooncogenes

Point mutations in ras genes have been found in a large number and wide variety of human tumors. These oncogenic Ras mutants are locked in an active GTP-bound state that leads to a constitutive and deregulated activation of Ras function. The dogma that ras oncogenes are dominant, whereby the mutation of a single allele in a cell will predispose the host cell to transformation regardless of the presence of the normal allele, is being challenged. We have seen that increasing amounts of Ras protooncogenes are able to inhibit the activity of the N-Ras oncogene in the activation of Elk in NIH 3T3 cells and in the formation of foci. We have been able to determine that the inhibitory effect is by competition between Ras protooncogenes and the N-Ras oncogene that occurs first at the effector level at the membranes, then at the processing level and lastly at the effector level in the cytosol. In addition, coexpression of the N-Ras protooncogene in thymic lymphomas induced by the N-Ras oncogene is associated with increased levels of p107, p130 and cyclin A and decreased levels of Rb. In the present report, we have shown that the N-Ras oncogene is not truly dominant over Ras protooncogenes and their competing activities might be depending on cellular context.     

Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto-oncogene p21 products

The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. This gene family mediates transformation via (1) a point-mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21. Group-specific, type-selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and proto-oncogene products, have been developed. Using purified recombinant ras p21 from Escherichia coli expressing the full-length T24 mutant human Harvey-ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E. coli expressing the point-mutated or proto-ras p21 and (2) mammalian cell lines of human and murine origin. Two of the RIAs developed can be termed group-specific in that they have the ability to detect the point-mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type-selective, since it detects an antigenic determinant located primarily on the Harvey ras p21. All 3 RIAs are interspecies-specific since they are able to detect ras p21 in rodent as well as human cells. The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression. These assays, used in conjunction with specific cDNA probes to identify specific ras proto-oncogenes or point-mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and transformation.     

Enhanced invasive properties of rat embryo fibroblasts transformed by adenovirus E1A mutants with deletions in the carboxy-terminal exon.

E1A genes deficient in the carboxy-terminal exon can cooperate with activated ras oncogenes to induce transformation of rat embryo fibroblasts. However, the resulting transformed foci show a distinct appearance characterized by a decreased adhesion of the cells to the substrate. Here, we demonstrate that cell lines derived from foci showing the variant morphology are defective in down-regulation of stromelysin 1 metalloprotease expression and show an increased invasive propensity compared with cells transformed by wild-type E1A. The altered focus morphology, the high invasive propensity and the elevated stromelysin 1 expression were abrogated by glucocorticoid treatment. Our results show that E1A functions necessary for transformation and inhibition of invasive properties may be separated, and indicate that a 23 amino acid serine/threonine-rich region within the E1A carboxy-terminal exon is required for efficient repression of metalloprotease expression in transformed cells.     

Genetic analysis of invasion and metastasis

Metastasis formation is a multistep process that probably requires a complex interplay of a large and heterogeneous group of genes, including genes involved in cellular resistance to immunorejection and genes controlling the invasive potential of cells. Transfection experiments have shown that oncogenes of the ras gene family as well as oncogenes of the kinase group are able to induce invasive and metastatic properties in non-transformed cells as well as in tumorigenic, but non-metastatic, cells. However, these findings are not in agreement with observations on spontaneous human tumours in which no correlation was found between activation or increased expression of ras genes and the invasive and metastatic properties of these cells. Further studies have indicated that in particular cell types nuclear oncogenes like N-myc and adenovirus derived E1A may influence metastasis formation by trans-regulation of other genes, including class I genes of the major histocompatibility complex and genes coding for proteolytic enzymes. The many efforts to identify additional invasion and metastasis associated genes by transfection of high molecular weight metastatic tumour DNA met with little success. Somatic cell fusion studies, however, indicate that such genes exist and that one or more of them are located on human chromosome 7.     

Roles for the coactivators CBP and p300 and the APC/C E3 ubiquitin ligase in E1A-dependent cell transformation

Adenovirus early region 1A (E1A) possesses potent transforming activity when expressed in concert with activated ras or E1B genes in in vitro tissue culture systems such as embryonic human retinal neuroepithelial cells or embryonic rodent epithelial and fibroblast cells. Early region 1A has thus been used extensively and very effectively as a tool to determine the molecular mechanisms that underlie the basis of cellular transformation. In this regard, roles for the E1A-binding proteins pRb, p107, p130, cyclic AMP response element-binding protein (CBP)/p300, p400, TRRAP and CtBP in cellular transformation have been established. However, the mechanisms by which E1A promotes transformation through interaction with these partner proteins are not fully delineated. In this review, we focus on recent advances in our understanding of CBP/p300 function, particularly with regard to its relationship to the anaphase-promoting complex/cyclosome E3 ubiquitin ligase, which has recently been shown to interact and affect the activity of CBP/p300 through interaction domains that are evolutionarily conserved in E1A.     

Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells

Previous studies have demonstrated that the HPV-16 E7 gene product encodes the major transforming activity of the virus in rodent cell systems. In this study we have generated a series of point mutations affecting the region of HPV-16 E7, which shows homology to adenovirus E1a conserved domain (CD)1. In conjunction with previously described mutants in the region of E7 with similarity to E1a CD2 and SV40 LT, we have investigated three known activities of the E7 protein; transformation, association with the cellular RB protein and induction of cellular DNA synthesis. The results show that RB binding correlates with the ability of E7 to induce cellular DNA synthesis and mediate cell transformation. In addition an unidentified function of E7, which is necessary for transformation of NIH3T3 cells, but does not affect RB binding or the ability to induce cellular DNA synthesis, has also been demonstrated. This study therefore identifies two separate regions of the E7 gene necessary for transformation of established cells. One of these, in the region of E7 which shows similarity to E1a CD2 and LT, is required for RB binding and DNA synthesis. The other region important for transformation, in the N-terminus of E7, is separable from the RB binding/DNA synthesis function.     

Identification and quantification of a carcinogen-induced molecular initiation event in cell transformation.

All transformed foci of Balb/c 3T3 clone A31-1-1 cells induced by 7,12-dimethylbenz[a]anthracene (DMBA) (42 out of 42 examined) contained an A to T transversion at codon 61 (A182 to T) of the Ki-ras gene. The transformants induced by other carcinogens tested did not contain such a mutation, except one out of nine 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced transformed foci. Thus, we hypothesized that this mutation is a specific DMBA-induced initiating event in Balb/c 3T3 cell transformation and we have measured its frequency of induction before transformation occurs, employing our recently developed method. Such mutations can be detected in the cell population as early as 3 days after exposure to DMBA. The same mutation was also detected in the Ha-ras gene. No detectable level (< 10(-6) of these mutations was induced by other carcinogens tested. The mutation frequency of the Ha-ras gene reached a plateau after 1 week's exposure, but that of the Ki-ras gene continued to increase. These results suggest that the A182 to T mutation of the Ki-ras gene, but not that of the Ha-ras gene, contributes to morphological transformation of Balb/c 3T3 cells. We have demonstrated that the level of expression of ras genes determines the rate of recruitment of cells into transformation. Quantitative analysis of the frequencies of ras gene mutations (initiation) and of transformation suggests that about 25% of those cells with the Ki-ras mutation were recruited into the full transformation process and that, in the presence of the tumor promoter TPA, about 56% of them completed morphological cell transformation.     

Involvement of the ras genes in female genital tract cancer

Human carcinogenesis is a multistep process involving complicated genetic events in which several onco-genes and oncosuppressor genes are implicated. The role of ras oncogenes in cellular transformation and apoptosis has been extensively examined and the dual role of ras as oncogene and oncosuppressor gene has been supported. Activation of ras occurs either by genomic alterations such as point mutations or by modulation of Ras protein expression. Many molecular and immunohistochemical studies have focused on ras activation in a wide range of human tumours. In this review, we summarize available information regarding the genomic and expression alterations of ras oncogenes in cervical, endometrial and ovarian cancer. Gynecological malignancies represent some of the most well- studied types of human cancer concerning ras activation and its possible use in clinical practice.     

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.     

Tumorigenic Transformation of Primary Rat Embryonal Fibroblasts by Human Papillomavirus Type 8 E7 Gene in Collaboration with the Activated H-ras Gene

Particular types of human papillomavirus (HPV) are associated with skin cancer of epidermo-dysplasia verruciformis (EV) patients. Here, we show, for the first time, that the E7 gene of EV-associated HPV8 possesses a potential oncogenic transforming ability. The HPV8 E7 open reading frame (ORF) and the HPV16 E7 ORF were cloned under the SV40 promoter/enhancer to construct recombinant plasmids pcD2-8E7 and pcD2-16E7, respectively. Transfection of primary rat embryonal flbroblasts having an activated H- ras gene revealed that pcD2-8E7 as well as pcD2-16E7 induced transformation of cells In G418-resistant colonies at an efficiency of 12.3% and 42.9%, respectively. The resulting transformed cell lines induced by pcD2-8E7 and activated H- ras were tumorigenic when injected into syngeneic immunocompetent rats. The potential tumorigenicity of HPV8 E7 seemed to be higher than that of HPV16 E7.     

N-methyl-N-nitrosourea-induced transformation of rat urothelial cells in vitro is not mediated by activation of ras oncogenes

Adult rat urothelial cells were transformed in vitro following treatment with a single dose of N-methyl-N-nitrosourea (MNU) or MNU treatment followed by promotion with sodium saccharin. This in vitro transformation process involves multiple steps: slow-growing 'pre-neoplastic' epithelial foci are induced 70-100 days after MNU treatment and from such foci rapidly proliferating immortal cell lines were established, some of which became tumorigenic after a further latent period. A series of epithelial cell lines and a single fibroblast cell line established in this way were analysed for the presence of transforming genes by DNA transfection into NIH3T3 cells. None of the epithelial cell lines induced foci in a focus formation assay. The single non-epithelial line induced foci and was found to contain an activated c-Ki-ras gene with a G----A transition in codon 12. To assay for the possible presence of transforming genes which were not active in a focus formation assay, two of the epithelial lines were analysed further by co-transfection with a dominant selectable marker, followed by selection and inoculation into nude mice. No tumours were induced within the latent period for tumour production by control cells transfected with NIH3T3 cell DNA (40-60 days). These results suggest that there is cell type specificity for oncogene activation during in vitro rat bladder transformation initiated by a single carcinogen and that ras gene activation is not a necessary step in urothelial transformation in vitro.