Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene

Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated c-rasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fos/FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express gamma-glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since gamma-glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated c-rasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.     

Expression of intermediate filament proteins by normal and transformed mouse epidermal-keratinocytes in culture

The aberrant expression of embryonic simple epithelial keratins K8 and K18 has been previously detected in chemically-induced mouse skin carcinomas and in cultured epidermal keratinocytes containing H-ras oncogenes. In this study, we report that cell lines derived from benign papillomas do not generally synthesize simple epithelial keratins, regardless of the presence of H-ras gene alterations. These results reinforce our previous suggestion that the expression of these keratins during mouse epidermal carcinogenesis is a marker of malignant transformation. The comparison of the two-dimensional electrophoresis intermediate filament protein profiles of transformed keratinocytes in culture, reveal that during in vitro progression to the malignant phenotype increased synthesis of simple epithelial keratins is associated to a general disturbance on the expression of cytoskeletal proteins.     

Cooperation between mutant p53 and the ras, raf, erbb-2 and fgf-3 oncogenes for transformation of mammary epithelial-cells

The murine mammary epithelial cells HC11 were used as a model to examine cooperation between mutated p53 and activated oncogenes for cell growth and transformation. These cells lack wild-type (wt) p53 and their proliferation in monolayer is inhibited by reitroduction of wt p53. Expression of the ras, raf, erbB-2 and fgf-3 (former int-2) oncogenes in HC11 cells leads to their growth in soft-agar, a parameter of cell transformation. Clonogenicity in soft-agar of the ras, raf and erbB-2 transformed cells was inhibited by a temperature-sensitive (ts) p53 at 32 degrees C, when the ts p53 protein is wt. Thus these oncogenes act synergistically with mutant p53 to induce anchorage-independent growth. Proliferation in monolayer of erbB-2, but not ras, raf, or fgf-3, transformed cells was retarded by ts p53 at 32 degrees C. Thus, ras, raf and fgf-3 oncogenes can partly or completely overcome the proliferation inhibitory function of wt p53, while erbB-2 cannot. These data indicate that specific oncogenes can distinctly cooperate with p53 for growth and transformation of mammary cells.     

p19/Arf and p53 suppress sentinel lymph node lymphangiogenesis and carcinoma metastasis

The ability of tumor cells to metastasize is increasingly viewed as an interaction between the primary tumor and host tissues. Deletion of the p19/Arf or p53 tumor suppressor genes accelerates malignant progression and metastatic spread of 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced squamous cell carcinomas, providing a model system to address mechanisms of metastasis. Here, we show that benign pre-metastatic papillomas from wild-type mice trigger lymphangiogenesis within draining lymph nodes, whereas there is no growth of primary tumor lymphatic vessels. Lymph node lymphangiogenesis is greatly accelerated in papilloma-bearing p19/Arf- or p53-deficient mice, which coincides with the greater propensity of these tumors to progress to carcinomas and to metastasize. The extent of accumulation of B cells within the tumor-draining lymph nodes of wild-type mice predicted the level of lymph node lymphangiogenesis and metastatic potential. Arf or p53 deficiency strongly accelerated lymph node immune cell accumulation, in a manner that was associated with the extent of lymph node lymphatic sinus growth. This immune cell accumulation and lymph node lymphangiogenesis phenotype identifies host anti-tumor responses that could drive metastatic spread of cancers via the lymphatics.     

Selective cytotoxic effect of tamoxifen on epithelial and fibroblastic cells transformed by different oncogenes

To study the correlation between oncogenes and the cytotoxic effect of tamoxifen in estrogen negative (ER) cells, we transformed mouse keratinocyte and fibroblastic cell lines with several oncogenes and studied cell viability, thymidine incorporation and PKC levels. We show that v-myc and v-H-ras oncogenes increase sensitivity in both cell types and that Neu and mutant p53 also increase sensitivity to tamoxifen, more significantly in the epithelial cells. Conversely, transformation with adenovirus E1a oncogene induces resistance to tamoxifen in both cell types. These results indicate that tamoxifen may be effective in different kinds of malignant cells depending on the oncogenic alterations present in the cells.     

Mammary epithelial cell transformation: insights from cell culture and mouse models

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16^INK4a, p21, p27 and p57), p14^ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.     

Protection against malignant conversion in SENCAR mouse skin by all Trans retinoic acid: Inhibition of the ras p21-processing enzyme farnesyltransferase and Ha-ras p21 membrane localization

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01–0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene. © 1996 Wiley-Liss, Inc.     

Oncogenes and metastatic progression

It is now established that ras oncogenes can induce metastatic characteristics in primary diploid fibroblasts, nonsenescing fibroblasts and nonmetastasizing tumors. The issue of whether ras is directly involved in maintaining the metastatic phenotype through the expression and action of its gene product has been examined by analyzing the relationship to ras expression and to the production of the p21 ras-GTP complex, which is thought to mediate ras-transforming activity. While these expression and mutation studies support the idea that p21 ras directly regulates metastasis formation, it is also evident that there are many examples of human and murine cancers which show no differences in ras expression in primary and metastatic tumor cells. This may be partially explained by the ability of protein kinase-encoding oncogenes to also induce metastatic potential. In addition, the ability of ras to induce metastasis may be dependent on the regulation of its activity by other genes. Furthermore, transformation does not occur as an isolated genetic event, but is rather the result of interaction of two or more oncogenes. We suggest that the nature of these gene interactions will ultimately determine whether a cell is a benign transformant or a malignant and metastatic cancer.     

Immunological prevention of a multigene cancer syndrome

Vaccines effectively prevent the onset of tumors in transgenic mice carrying activated oncogenes; however, human tumors are caused by combined alterations in oncogenes and oncosuppressor genes. We evaluated the impact of prophylactic vaccines in HER-2/neu transgenic, p53 wild-type/null mice that succumb to an aggressive cancer syndrome comprising mammary and salivary gland carcinomas and rhabdomyosarcoma. A vaccine made of allogeneic mammary carcinoma cells expressing HER-2/neu and interleukin 12 afforded long-term protection from tumor onset. Tumor prevention was mediated by T cell-derived cytokines, in particular gamma-interferon, and by anti-HER-2/neu antibodies. HER-2/neu expression was inhibited in target tissues of vaccinated mice, and somatic loss of the wild-type p53 allele did not occur. A highly effective vaccine against a single oncoprotein induced a powerful immune response that arrested multistep carcinogenesis in distinct target tissues.     

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.     

Induction of ovarian cancer by defined multiple genetic changes in a mouse model system

We have developed a mouse model for ovarian carcinoma by using an avian retroviral gene delivery technique for the introduction of multiple genes into somatic ovarian cells of adult mice. Ovarian cells from transgenic mice engineered to express the gene encoding the avian receptor TVA were efficiently infected in vitro with multiple vectors carrying coding sequences for oncogenes and marker genes. When target cells were derived from TVA transgenic mice deficient for p53, the addition of any two of the oncogenes c-myc, K-ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected at subcutaneous, intraperitoneal, or ovarian sites. We demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells. The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread.     

Sequential studies of skin tumorigenesis in PGK mosaic mice: the effect of repeated exposure to a carcinogen on regressed mouse skin papillomas

Most mouse skin papillomas developing after 7,12-diinethylbenz[a]anthracene(DMBA) initiation followed by repeated 12-O-tetradecanoylphorbol-13-acetate(TPA) promotion are promoter-dependent; termination of promotionresults in their regression. Previous evidence, from mappingthe locations of papillomas and using the X-chromosome-linkedphosphoglycerate kinase cell markers, shows that regressionof promoter-dependent papillomas is permanent. Exposure to anothercourse of TPA promotion was found not to induce regenerationin the regressed papillomas. To determine whether repeated exposureto a carcinogen causes regeneration of regressed papilomas,the effects of weekly applications of low doses of DMBA weretested. The results reported here indicate that regressed promoter-dependentpapillomas do not regenerate when exposed repeatedly to DMBA.However, the treatment with DMBA induced many new tumors mostof which were promoter-independent papillomas or malignant carcinomas.These findings provide further support for the conclusion thattermination of promotion leads to permanent regression of mostpromoter-dependent mouse skin papillomas.     

Protection against induction of mouse skin papillomas with low and high risk of conversion to malignancy by green tea polyphenols

We earlier showed that a polyphenolic fraction isolated from green tea (GTP) affords protection against tumor promotion and tumor progression in SENCAR mouse skin. The present study was designed to further evaluate the protective effect of GTP against the induction and subsequent progression of papillomas to squamous cell carcinomas (SCCs) in experimental protocols where papillomas were developed with a low or high probability of their malignant conversion. Topical application of GTP (6 mg/animal) 30 min prior to that of 12-O-tetradecanoylphorbol-13- acetate (TPA) either once a week for 5 weeks (high risk TPA protocol) or once a week for 20 weeks (low risk TPA protocol) or mezerein (MEZ) twice a week for 20 weeks (high risk MEZ protocol) in 7,12- dimethylbenz[a]anthracene (DMBA)-initiated mouse skin resulted in significant protection against skin tumor promotion in terms of tumor incidence (32-60%), multiplicity (49-63%) and tumor volume/mouse (73- 90%) at the termination of the experiment at 20 weeks. In three separate malignant progression experiments when papilloma yield in DMBA- initiated and TPA or MEZ promoted low and high risk protocols was stabilized at 20 weeks, animals were divided into two subgroups. These animals were either topically treated twice weekly with acetone (0.2 ml/animal, spontaneous malignant conversion group) or with GTP (6 mg/animal in 0.2 ml acetone) for an additional period of 31 weeks. During these treatment regimens, all suspected carcinomas were recorded and each one was verified histopathologically either at the time when tumor-bearing mouse died/moribund or at the termination of the experiment at 51 weeks. GTP resulted in significant protection against the malignant conversion of papillomas to SCC in all the protocols employed. At the termination of the experiment at 51 weeks, these protective effects were evident in terms of mice with carcinomas (35- 41%), carcinomas per mouse (47-55%) and percent malignant conversion of papillomas to carcinomas (47-58%). The kinetics of malignant conversion suggest that a subset of papillomas formed in the early phase of tumor promotion in all the protocols had a higher probability of malignant conversion into SCCs because all the positive control groups (acetone treated) produced nearly the same number of carcinomas (33-38 in a group of 20 animals) at the end of the progression period. In the GTP- treated group of animals the number of carcinomas formed was less (14- 20 in a group of 20 animals), which shows the ability of GTP to protect against the malignant conversion of papillomas of higher probability of malignant conversion to SCCs. The results of this study suggest that irrespective of the risk involved, GTP may be highly useful in affording protection against skin cancer risk.      

Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis

Ras genes are the most frequently mutated oncogenes in human cancer. However, the contribution of ras to tumor initiation still is unclear because ras expression in primary cells can cause cell cycle arrest and even cell death by apoptosis. Furthermore, when expressed in the epidermis of mice, mutant ras promotes the formation of benign papillomas, only few of which will progress into carcinomas. However, in these cases, ras-transgene expression often is restricted to suprabasal or follicular epithelial cells that may lack self-renewal capacity. Thus, it still is conceivable that expression of active ras in other epithelial compartments may exert a distinct ability to promote malignant progression. To address this possibility, transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium, which includes the epithelial stem cells, were engineered and crossed with mice expressing the K-ras(G12D) oncogene under the control of tet-regulated responsive elements. On doxycycline administration, proliferative lesions ranging from hyperplasias, papillomas, and dysplasias to metastatic carcinomas developed in squamous epithelia of the skin, oral mucosa, salivary glands, tongue, esophagus, forestomach, and uterine cervix within just 10 to 20 days. The most noticeable lesions were invasive squamous carcinomas of the skin and oral mucosa. These findings suggest that the expression of oncogenes in an epithelial compartment that includes the stem cells may be sufficient to promote squamous carcinogenesis. They also provide a molecularly defined conditional animal model system in which the mechanisms responsible for cancer initiation, maintenance, and metastatic spread can be readily investigated.     

Heterogeneity of lung cancer cells with respect to the amplification and rearrangement of myc family oncogenes

Seventy lung tumors from 53 patients were analysed for alterations of myc family oncogenes, c-myc, N-myc and L-myc, to evaluate when activation of these genes occurs during tumor development. The 53 cases were 17 small cell carcinomas (SCCs), 18 adenocarcinomas, 12 squamous cell carcinomas (SqCs), 4 large cell carcinomas and 2 adenosquamous carcinomas. Either N-myc or L-myc was amplified in 4 of the 17 (one N-myc and 3 L-myc) SCCs (24%), while c-myc was amplified in 3 of the 12 SqCs (25%). In one SCC, amplification of N-myc was found in the primary tumor, a pulmonary hilar lymph node metastasis and a pleural metastasis, but not in a liver metastasis or a para-aortic lymph node metastasis. In one SqC, c-myc was amplified in a pleural metastasis and a lymph node metastasis, but not in the primary tumor. In 2 cases of SCCs, amplification or rearrangement of c-myc was detected only in the cell lines, but not in the original tumors taken from the same individuals. These results indicate that tumor cells were heterogeneous for amplification and rearrangement of myc family oncogenes, and suggest that activation of these oncogenes in SCCs and SqCs occurs not at the time of malignant transformation but during tumor progression.     

Loss of heterozygosity and mutational alterations of the p53 gene in skin tumours of interspecific hybrid mice.

Functional alterations or loss of tumor-suppressor genes are an important feature of neoplastic progression in humans. The employment of suitable animal model systems would greatly facilitate the detection and manipulation of such genes. We describe here an experimental approach to this problem based on the analysis of skin tumors induced in F1 hybrids between Mus musculus and Mus spretus mice. The results show that loss of heterozygosity on chromosome 11 occurred in 4/13 mouse skin carcinomas, but not in premalignant papillomas. Since the murine p53 gene is located on this chromosome, immunoprecipitation and DNA-sequencing studies were carried out on tumorigenic cell lines and primary tumor DNA respectively to determine the status of p53 alleles. These studies revealed the presence of p53 mutations, both frameshifts and missense, some of which are identical to those found in human tumors. Loss of normal p53 function is found in well-differentiated squamous-cell carcinomas and thus does not appear to be directly responsible for further progression to an undifferentiated spindle cell phenotype.     

Sequential trisomization of chromosomes 6 and 7 in mouse skin premalignant lesions

Using a direct cytogenetic technique, we identified a nonrandom trisomy of chromosome 6 in 12 of 12 aneuploid mouse skin papillomas and in 10 of 11 squamous cell carcinomas induced by chemical carcinogenesis. The second most common abnormality observed was trisomy of chromosome 7 found in most dysplastic papillomas and in 10 of 11 carcinomas. The two trisomies were the only abnormalities found in all aneuploid papillomas and in several carcinomas. Mutation at codon 61 of the Ha-ras gene, which resides on chromosome 7, was also a common feature of the tumors sampled. Extensive homology exists between mouse chromosome 6 and human chromosome 7, the trisomy of which was recently suggested as a primary cytogenetic event in several human epithelial cancers. We propose a multistep model of tumor progression in which a sequence of specific nonrandom chromosomal abnormalities appear to be required for malignant transformation.