Malignant properties of sublines selected from a human bladder cancer cell line that contains an activated c-Ha-ras oncogene

The human bladder cancer cell line MGH-U1 (also designated T-24 or EJ) contains an activated c-Ha-ras oncogene, which is amplified as compared to normal human fibroblasts. We have generated sublines from the MGH-U1 cell line: the MGH-U1/OCI subline was generated by dissociating spheroids formed from MGH-U1 cells; the U1-m/F1 and OCI-m/F1 were generated by in vivo passage of experimental lung metastases formed after i.v. injection of MGH-U1 and MGH-U1/OCI lines into immune-deprived mice; the U1/t subline was generated by in vivo passage of i.m. tumors formed from MGH-U1 cells. All sublines formed tumors in immune-deprived mice from smaller i.m. inocula than the parent line, and the U1-m/F1 subline generated more spontaneous metastases in lungs. Lung colony forming efficiency after i.v. injections of cells into similar mice was also greater for the sublines than for the parent MGH-U1 cells. The U1-m/F1 and OCI-m/F1 were the most tumorigenic lines. Early passages of the MGH-U1/OCI subline showed the presence of double minute chromosomes, and amplification and increased expression of the c-Ha-ras oncogene as compared to the parental cell line. These changes were not present in later cultures of MGH-U1/OCI cells, and no consistent difference in the levels of gene amplification or expression between the parent line and the sublines was found. Thus the content and expression of the activated c-Ha-ras oncogene does not correlate with malignant properties of the sublines.     

Fgf10 is an oncogene activated by MMTV insertional mutagenesis in mouse mammary tumors and overexpressed in a subset of human breast carcinomas

Mouse mammary tumor virus (MMTV) infection causes a high incidence of murine mammary carcinomas by insertion of its proviral DNA in the genome of mammary epithelial cells. Retroviral insertion can activate flanking proto-oncogenes by a process called insertional mutagenesis. By sequencing the DNA adjacent to MMTV proviral insertions in mammary tumors from BALB/c mice infected with C3H-MMTV, we have found a common MMTV insertion site in the Fgf10 locus. RT-PCR studies showed that Fgf10 is expressed only in those tumors harboring a MMTV proviral insertion in this locus, suggesting that Fgf10 is a proto-oncogene. The oncogenicity of Fgf10 was evaluated in vivo by subcutaneous transplantation of retrovirally transduced HC11 mammary epithelial cells into BALB/c mice. Highly vascularized invasive subcutaneous tumors developed indicating that Fgf10 can act as an oncogene. A survey of primary human breast carcinomas revealed strongly elevated Fgf10 mRNA levels in approximately 10% of the tumors tested, suggesting that Fgf10 may also be involved in oncogenicity of a subset of human breast cancers.     

Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene

Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock has been shown to increase the frequency of DNA-mediated gene transfer to mammalian cells as compared with the frequency obtained with calcium phosphate transfection. We have successfully adapted this procedure for use with human epidermal keratinocytes. Non-tumorigenic human epidermal epithelial cells immortalized by SV40 tumor antigen were neoplastically transfected, using Polybrene at a concentration of 10 micrograms ml-1, followed by a 4 min shock, with 30% DMSO, with a plasmid carrying the activated H-ras gene from the EJ bladder carcinoma cell line. The transfected cells showed morphological alterations and induced carcinomas when transplanted into nude mice. They contained integrated copies of the transfected H-ras gene and expressed high levels of the p21 protein. Polybrene-induced DNA transfection, therefore, offers the opportunity to transfer genes effectively into human epidermal keratinocytes and should accelerate the study of the interaction between oncogenes and human epithelial cells. This study appears to represent the first neoplastic conversion of nontumorigenic, immortalized human epidermal keratinocytes by an activated human oncogene.     

Enhancement of benzo(a)pyrene metabolism mediated by retinol acetate in cultured human bronchial epithelial-cells

As suggested by animal studies and human epidemiological data, retinoids possess significant cancer chemopreventive activity. Although the majority of studies in this area have focused on the ability of retinoids to prevent the promotion or progression of carcinogenesis, a significant amount of data suggest retinoids can alter initiation events. In the current report, we have evaluated the potential of retinol acetate to modulate benzo(a)pyrene metabolism in low-passage human bronchial epithelial cells in monolayer cultures, Of 16 different cell cultures, benzo(a)pyrene metabolism was increased in 14, decreased in one, and unchanged in one, when retinol acetate was added to the media, In a preliminary study with one of the cell cultures in which retinol acetate significantly enhanced benzo(a)pyrene metabolism, binding of carcinogen metabolites to;DNA was unaffected, Since retinoids are known cancer chemopreventive agents and carcinogen binding to DNA is the key event in the initiation of carcinogenesis, these results suggest that retinoids may decrease carcinogenic risk by increasing the detoxification of procarcinogens such as benzo(a)pyrene ina manner that does not yield a concomitant increase in damage to critical cellular targets such as DNA.     

Influence of diet on mammary cancer in transgenic mice bearing an oncogene expressed in mammary tissue

Breast cancer is one of the most commoncancers in women. The laboratory rat treated withstrong carcinogen is the most commonly used animalmodel for study of breast cancer. Transgenic mouselines with homologues of human breast cancer oncogeneshave been developed. The transgenic mouse line TG.NKwith c-neu, the human breast cancer oncogene homologueof erbB2, was evaluated to determine its suitabilityfor study of intervention strategies to delay/prevent thedevelopment of breast cancer. There were no palpablemammary tumor masses up to 22-weeks of age,and almost all mice fed a purified dietdeveloped palpable mammary tumors by 28-weeks of age.Nonpurified diets decreased the incidence and multiplicity, anddelayed the development of mammary tumors as comparedto a purified diet. Increasing the fiber contentof nonpurified diet decreased the tumor incidence further.There is approximately a 19-week interval between weaningand development of palpable mammary masses to evaluateintervention strategies to delay or prevent the developmentof mammary cancer in the TG.NK mouse model.Fiber from nonpurified cereal ingredients appears to behighly beneficial in delaying the development of mammarycancer in TG.NK mice, and this observation isin agreement with human epidemiological findings. Therefore, theTG.NK transgenic mouse with oncogene c-neu (erbB2), appearsto be a useful animal model for evaluationof dietary intervention strategies.     

Metastatic ability and expression of c-fos oncogene in cell clones of a spontaneous rat mammary tumor

It was found that cell clones c1-2, cl-2r, c1-3, c1-4 and c1-6 of a spontaneous rat mammary tumor, c-SST-2, exhibit different degrees of metastatic ability: c1-2, c1-3, c1-6 were highly metastatic, while c1-2r and c1-4 were weakly metastatic. The expression of several oncogenes in these clones was examined. The amounts of myc mRNA in the clones were nearly the same. Expression of N-ras mRNA was higher in c1-2r and c1-4 than in c1-2, c1-3 and c1-6. On the other hand, the amounts of fos mRNA in the weakly metastatic clones were markedly lower than those in the highly metastatic clones. These results suggest that fos oncogene plays a role in the high metastatic ability of c-SST-2.     

Causal role for an activated N-ras oncogene in the induction of tumorigenicity acquired by a human cell line

ras oncogenes have been found in approximately 15% of the human tumors analyzed. However, a causal role for these genes in the tumorigenesis of human cells has yet to be shown. Tumorigenic late-passage PA-1 human teratocarcinoma cells (E-PA-1) contain an activated N-ras gene. In this report evidence is presented that nontumorigenic early passage revertant PA-1 cells (E-PA-1) contain only the germ-line protooncogene. Introduction by gene transfer of the activated L-PA-1 oncogene induces E-PA-1 cells to form tumors, suggesting that the activated N-ras oncogene has a causal role in the tumorigenesis of these cells.     

Hyperplasia of mouse mammary epithelium induced by expression of the Wnt-1 (int-1) oncogene in reconstituted mammary gland.

We have expressed the Wnt-1 (formerly int-1) oncogene in Balb/c mouse mammary epithelium in vivo, using a tissue reconstitution method in which primary cultures of mammary epithelial cells are infected with a retrovirus vector and then transplanted into mouse mammary fat pads from which the natural epithelium has been removed. Transplants carrying the Wnt-1 gene grew in a hyperplastic pattern, the duct epithelium showing abundant fine side-branches, but without development of clusters of alveoli. The hyperplasias were similar, but not identical, to transplants of normal epithelium in a mid-pregnant host. Transplants of epithelium that expressed Wnt-1 into mammary fat pads of male or ovariectomized females grew to form a similar three-dimensional pattern, but the extent of growth, and so presumably the rate of growth, was slower than in intact females, and there were no terminal end buds at the edges of the outgrowths. Thus, although Wnt-1 may enhance growth of epithelium in the male or ovariectomized-female environment, it does not restore the major mode of growth in the intact female, the extension of major ducts from terminal end buds. Normal epithelium showed no change in morphology when in close proximity to hyperplasia induced by Wnt-1, confirming the limited range of diffusion of Wnt-1 protein in vivo. Our results are consistent with the hypothesis that Wnt-1 acts principally by mimicking the signal that causes ducts to develop side-branches in pregnancy.     

Histopathology of salivary and mammary gland tumors in transgenic mice expressing a human Ha-ras oncogene

Mutated ras genes are powerful transforming agents in vitro and are found in a wide variety of human tumors in vivo. We characterized the histopathology and p21 protein expression associated with tumorigenesis in line 69 transgenic mice carrying an activated, human c-Ha-ras gene on the Y-chromosome (A. C. Andres, C. A. Schonenberger, B. Groner, L. Hennighausen, M. LeMeur, and P. Gerlinger, Proc. Natl. Acad. Sci. USA, 84: 1299-1303, 1987). Male mice developed salivary and/or mammary gland tumors. The salivary tumors were adenosquamous carcinomas arising from serous areas of the submandibular gland. They characteristically exhibited densely packed cords and sheets of moderately anaplastic cells. Tumorigenic tissue had a high mitotic index, and all tumor-bearing animals had an ongoing inflammatory response as evidenced by extensive immune cell infiltration of affected tissue. Half of the mammary gland tumors were adenosquamous carcinomas with multiple foci of squamous metaplasia, while the rest were adenocarcinomas containing glandular tissue. Most tumors had a high mitotic index, and abnormal mitotic figures were common. All tumors produced p21 ras, as confirmed by immunohistochemistry and Western blots. Both tumor types expressed elevated levels of p21 protein. Microscopic lung metastases were present in 5 of 35 animals (14%). Our results suggest that this transgenic mouse will provide a useful model for testing therapies directed against ras-associated tumorigenesis.     

Expression of an activated Notch4(int-3) oncoprotein disrupts morphogenesis and induces an invasive phenotype in mammary epithelial cells in vitro

The protein encoded by the Notch4 gene is a member of the Notch/lin-12 family of transmembrane receptor proteins, which have been shown to control cell fate determination and cell differentiation in a wide variety of organisms. Expression of Notch4(int-3), a truncated form of Notch4 having most of its extracellular domain deleted, as a transgene in mice induces the formation of poorly differentiated mammary carcinomas. To establish whether Notch4(int-3) has the capacity of subverting normal epithelial architecture, we assessed the effect of Notch4(int-3) expression on the in vitro morphogenetic properties of TAC-2 mammary epithelial cells. When grown in three-dimensional collagen gels in the presence of hydrocortisone, both wild-type and LacZ-transfected TAC-2 cells formed alveolar-like structures composed of polarized epithelial cells surrounding a central lumen. In contrast, TAC-2 cells programmed to express Notch4(int-3) formed compact cell aggregates devoid of tissue-specific organization. In addition, when grown on the surface of a collagen gel, Notch4(int-3)-expressing TAC-2 cells invaded the underlying matrix, whereas TAC-2 LacZ cells remained strictly confined to the gel surface. Expression of Notch4(int-3) in TAC-2 cells also disrupted contact-inhibition of cell proliferation, resulting in cell multilayering. Our results suggest that the ability of Notch4(int-3) to subvert normal epithelial morphogenesis and to promote invasion of the extracellular matrix contributes significantly to its tumorigenic potential.     

Massive accumulation of neutral lipids in cells conditionally transformed by an activated H-ras oncogene

Phenotypic consequences of ras oncogene expression were studied in cells conditionally transformed by T24 H-ras and a temperature-sensitive SV40 large T antigen (tsA58). Previous studies have demonstrated that transformation of REF52 cells by ras and SV40 large T antigen requires continuous T antigen expression. Thus, tsA58/T24 H-ras transformants ceased growing when transferred to a restrictive temperature for T antigen expression. Inhibition of cell growth was accompanied by massive accumulations of cholesterol esters, triglycerides and a third lipid species, identified as glycerol ethers on the basis of mobility on TLC. Cholesterol esters were derived from serum lipoproteins, and appeared to accumulate because LDL receptor expression and activity did not decline in growth arrested cells. Triglycerides and glycerol ethers were products of cell metabolism. The process lacked features characteristic of adipocyte differentiation, but may suggest mechanisms important in diseases, such as atherosclerosis, that involve abnormal accumulations of neutral lipids. Accumulating lipid species may also include metabolites induced by ras that accumulate in growth-arrested cells.     

H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer

Laser capture microdissection, polymerase chain reaction-restriction fragment length polymorphism analysis, and DNA sequencing was used to detect H-ras codon 12 and 13 mutations during the stages of mammary gland cancer development in rats exposed to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat. Ten oral doses of PhIP (75 mg/kg, p.o., once per day) were administered to adolescent female Sprague-Dawley rats and mammary glands examined histologically for intraductal proliferations (IDPs), carcinoma in situ and carcinomas 7-14 weeks later. Mammary gland epithelial cells from normal tissue and distinct lesions were collected from glass slides and analyzed for mutations. H-ras codon 12/13 mutations were detected in 73%, 75%, 100%, and 100% of normal mammary glands, IDPs, carcinoma in situ, and carcinoma, respectively, after PhIP treatment. The spectrum of activating mutations included G(35) to A or C base substitution mutations in codon 12, and G(37) to T or A base substitution mutations in codon 13. The spectrum of H-ras mutations was similar among normal mammary gland from PhIP treated rats, preneoplastic lesions, and carcinomas. Furthermore, the spectrum of mutations was consistent with the involvement of PhIP-guanine adduct formation. The results support the notion that mutations in H-ras codons 12 and 13 are largely PhIP-DNA adduct-induced and involved in the initiation and development of mammary gland cancer in rats exposed to PhIP.     

Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl)retinamide

Epithelial cells from non-cancerous mammary tissue in response to exposure to chemical carcinogens or transfection with oncogenes exhibit hyperproliferation and hyperplasia prior to the development of cancer. Aberrant proliferation may, therefore, represent a modifiable early occurring preneoplastic event that is susceptible to chemoprevention of carcinogenesis. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR), has exhibited preventive efficacy in several in vitro and in vivo breast cancer models, and represents a promising chemopreventive compound for clinical trials. Clinically relevant biochemical and cellular mechanisms responsible for the chemopreventive effects of HPR, however, are not fully understood. Experiments were performed on preneoplastic human mammary epithelial 184-B5/HER cells derived from reduction mammoplasty and initiated for tumorigenic transformation by overexpression of HER-2/neu oncogene, to examine whether HPR inhibits aberrant proliferation of these cells and to identify the possible mechanism(s) responsible for the inhibitory effects of HPR. Continuous 7-day treatment with HPR produced a dose-dependent, reversible growth inhibition. Long-term (21 day) treatment of 184-B5/HER cells with HPR inhibited anchorage-dependent colony formation by approximately 80% (P < 0.01) relative to that observed in the solvent control. A 24 h treatment with cytostatic 400 nM HPR produced a 25% increase (P = 0.01) in G0/G1 phase, and a 36% decrease (P = 0.01) in S phase of the cell cycle. HPR treatment also induced a 10-fold increase (P = 0.02) in the sub-G0 (apoptotic) peak that was down-regulated in the presence of the antioxidant N-acetyl-L-cysteine. Treatment with HPR resulted in a 30% reduction of cellular immunoreactivity to tyrosine kinase, whereas immunoreactivity to p185HER remained essentially unaltered. HPR exposure resulted in time-dependent increase in cellular metabolism of the retinoid as evidenced by increased formation of the inert metabolite N-(4-methoxyphenyl)-retinamide (MPR) and progressive increase in apoptosis. Thus, HPR-induced inhibition of aberrant proliferation may be caused, in part, by its ability to inhibit HER-2/neu-mediated proliferative signal transduction, retard cell cycle progression and upregulate cellular apoptosis.     

p53 levels in human mammary epithelial cells expressing wild-type and mutant human papillomavirus type 16 (HPV-16) E6 proteins

Human fibroblast cells must overcome both the M1 and the M2 stages of cellular senescence to immortalize, at which point cells almost always express telomerase activity. The human papillomavirus (HPV) oncoproteins, HPV-16 E6 and E7, can block the progression to senescence in fibroblasts by associations with p53 and pRb, respectively. Human mammary epithelial (HME) cells require only HPV-16 E6 to bypass M1, suggesting that pRb may not have a direct role in HME cells senescence. In the present report, we show that only wild-type HPV-16 E6 allows complete degradation of p53, immortalization and reactivation of telomerase activity in HME cells. These results suggest that the ability of HPV-16 wild-type and mutant E6 proteins to degrade p53 in intact HME cells and keratinocytes does not completely correlate with their ability to degrade p53 in a cell-free system. This discrepancy between in vitro and in vivo p53 degradation may be biologically significant and may provide insight into the susceptibility of certain human cells and tissues for reactivation of telomerase and immortalization.     

Complex agonist-like properties of ICI 182,780 (Faslodex) in human breast cancer cells that predominantly express progesterone receptor-B: implications for treatment resistance

ICI 182,780 (Faslodex), considered a pure anti-estrogen, is approved for treatment of post-menopausal breast cancer patients who fail to respond to tamoxifen therapy. We recently reported that, like mifepristone, ICI 182,780 exhibits anti-progestin activity, blocking the progestin-dependent increase in endogenous vascular endothelial growth factor (VEGF) mRNA and protein release. Some anti-progestins have partial agonist-like activity in breast cancer cells expressing high levels of progesterone receptor B (PRB). Our results show that ICI 182,780 can also induce reporter activity from a plasmid containing a simple progestin responsive element (PRE) in these cells. Using small interfering RNA, we determined that induction is dependent on the presence of PR, estrogen receptor and SRC-1. Regulation of more complex progestin-responsive promoters was context-dependent; induction was observed from the MMTV promoter but not from the VEGF promoter. In contrast, ICI 182,780 increased the release of angiogenically active VEGF from cells expressing elevated levels of PRB. This effect was dependent on the phosphatidylinositol-3 kinase and ERK/MAPK signaling pathways. We hypothesize that these agonist-like properties of ICI 182,780 (one genomic and one non-genomic) may contribute to the acquisition of drug resistance, suggesting that both anti-hormonal and anti-angiogenic treatment may be appropriate in these patients.