Pathologic progression of mammary carcinomas in a C3(1)/SV40 T/t-antigen transgenic rat model of human triple-negative and Her2-positive breast cancer.

The C3(1) component of the rat prostate steroid binding protein has been used to target expression of the SV40 T/t-antigen to the mammary epithelium of mice resulting in pre-neoplastic lesions that progress to invasive and metastatic cancer with molecular features of human basal-type breast cancer. However, there are major differences in the histologic architecture of the stromal and epithelial elements between the mouse and human mammary glands. The rat mammary gland is more enriched with epithelial and stromal components than the mouse and more closely resembles the cellular composition of the human gland. Additionally, existing rat models of mammary cancer are typically estrogen receptor positive and hormone responsive, unlike most genetically engineered mouse mammary cancer models. In an attempt to develop a mammary cancer model that might more closely resemble the pathology of human breast cancer, we generated a novel C3(1)/SV40 T/t-antigen transgenic rat model that developed progressive mammary lesions leading to highly invasive adenocarcinomas. However, aggressive tumor development prevented the establishment of transgenic lines. Characterization of the tumors revealed that they were primarily estrogen receptor and progesterone receptor negative, and either her2/neu positive or negative, resembling human triple-negative or Her2 positive breast cancer. Tumors expressed the basal marker K14, as well as the luminal marker K18, and were negative for smooth muscle actin. The triple negative phenotype has not been previously reported in a rat mammary cancer model. Further development of a C3(1)SV40 T/t-antigen based model could establish valuable transgenic rat lines that develop basal-type mammary tumors.

Persistent median artery: cadaveric study and review of the literature.

The persistent median artery (PMA) may compress the median nerve (MN) and may be a significant supply of blood to the hand. Two cases of unilateral PMA (4%) were detected during the dissection of 50 upper limbs. The first case was a 75-year-old, right-handed male who suffered from chronic pain in both upper limbs, especially the left side. A dissection of his left upper limb revealed a PMA piercing both the MN and the medial branch of the anterior interosseous nerve. This artery coursed distally, deep to the transverse carpal ligament (TCL), forming a median-ulnar pattern for the superficial palmar arch (SPA). The PMA was superficial to two nerves at the distal edge of the TCL; the extraligamentous recurrent thenar (RT) branch of the MN and the third common digital nerve (TCDN). The second case was from the left side of an 80-year-old female found to have a high origin of the radial artery with trifurcation of the latter into PMA, common interosseous, and ulnar arteries. The PMA passed deep to the TCL forming a radial-median-ulnar pattern of SPA. Both the transligamentous RT branch of the MN and the TCDN passed deep to the PMA inside the carpal tunnel, before the abnormal crossing of the latter nerve ventral to the SPA on its way to the digits. The relationships of the PMA to various MN branches may have important implications regarding the diagnosis and treatment of MN compressive neuropathies.

Electrogene therapy using endostatin, with or without suicide gene therapy, suppresses murine mammary tumor growth and metastasis.

Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of a plasmid vector containing either endostatin (pEndo) with or without a suicide gene (pHSVtk), pHSVtk alone or control vector once a week for 8 weeks. We applied electrogene transfer to the tumors after each injection and administered ganciclovir (GCV) to pHSVtk-transfected mice using an osmotic minipump. Anticancer efficacy was monitored using a variety of parameters, namely tumor volume, intratumoral microvessel density and DNA synthesis, number of mice with metastasis, and number of sites of metastasis per mouse. Tumor volume was significantly lower in all therapeutic groups, with the most effective growth suppression in the pEndo+pHSVtk/GCV group. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower only in the pEndo and pEndo+pHSVtk/GCV groups. All therapeutic groups showed significantly lower intratumor microvessel density and DNA synthesis. The pEndo and pEndo+pHSVtk/GCV groups also showed a significant reduction in the numbers of dilated lymphatic vessels containing intralumenal tumor cells. Our data suggest that endostatin electrogene therapy alone or in combination with pHSVtk/GCV suicide gene therapy is more beneficial than suicide gene therapy alone. The observed antimetastatic activity of endostatin may be of high clinical significance in the treatment of metastatic breast cancer.

Roles of urinary sodium ion concentration and pH in promotion by ascorbic acid of urinary bladder carcinogenesis in rats.

Since the sodium salt of ascorbic acid (AA) promoted two-stage urinary bladder carcinogenesis in rats, whereas AA itself did not, the roles of the urinary sodium ion concentration and pH on urinary bladder carcinogenesis were investigated. Male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks and then treated with basal diet containing 5% AA plus 3% sodium bicarbonate (NaHCO3), 5% AA, 3% NaHCO3 or 5% sodium L-ascorbate (SA), 5% SA plus 1% ammonium chloride (NH4Cl) or 1% NH4Cl, or no added chemical for 32 weeks. NaHCO3 significantly increased the induction of neoplastic and preneoplastic lesions of the urinary bladder. Like SA, AA plus NaHCO3 induced high incidences of neoplastic and preneoplastic lesions of the urinary bladder, whereas AA alone did not. NH4Cl reduced the promoting activity of SA in urinary bladder carcinogenesis. These results suggest important roles for urinary sodium ion concentration and pH in modulating urinary bladder carcinogenesis. Moreover, AA was found to act as a copromoter under conditions of increased urinary pH and sodium ion concentration.

Different modifying response of butylated hydroxyanisole, butylated hydroxytoluene, and other antioxidants in N,N-dibutylnitrosamine esophagus and forestomach carcinogenesis of rats.

The modifying effects of antioxidants were examined in a carcinogenesis system after N,N-dibutylnitrosamine treatment. Male F344 rats were given 0.05% N,N-dibutylnitrosamine in their drinking water for 4 wk and then treated with basal diet containing 2% butylated hydroxyanisole (BHA), 1% butylated hydroxytoluene (BHT) with 7 ppm vitamin K, 0.8% ethoxyquin, 5% sodium L-ascorbate, 5% sodium erythorbate, or no added chemical for 32 wk. BHA enhanced forestomach carcinogenesis but did not enhance esophageal carcinogenesis. BHT enhanced esophageal carcinogenesis but did not enhance forestomach carcinogenesis. Ethoxyquin significantly enhanced esophageal tumorigenesis. Neither esophageal nor forestomach carcinogenesis was affected by the other antioxidants evaluated. BHA significantly increased DNA synthesis of the forestomach epithelium, whereas BHT tended to increase that of the esophageal epithelium. Thus, BHA and BHT showed different modifying responses in carcinogenesis of the esophagus and forestomach.

Progression of prostatic intraepithelial neoplasia to invasive carcinoma in C3(1)/SV40 large T antigen transgenic mice: histopathological and molecular biological alterations.

The progression of prostatic intraepithelial neoplasia (PIN) to invasive prostate carcinoma has been analyzed in the C3(1)/T(AG) transgenic mouse model and appears very similar to the process proposed to occur in humans. PIN lesions in these transgenic mice histologically resemble those found in human PIN. Low-grade PIN was observed in the ventral and dorsolateral lobes at 2 months of age, whereas high-grade PIN was found in both lobes by 5 months of age. A progressive increase in the number of PIN lesions was observed with age. Prostate carcinomas, which appeared to arise from PIN lesions, were found by 7 months of age in the ventral lobe and 11 months of age in the dorsolateral lobe. Expression of T(AG) mRNA and protein in these lesions correlated with the development of PIN and carcinomas, as did the overexpression of p53 protein. Apoptosis levels were quite low in normal epithelial cells, moderate in low-grade PIN, and high in high-grade PIN and carcinomas. Levels of expression of proliferating cell nuclear antigen correlated with the degree of severity of the prostate lesions. Eighteen % of PIN lesions were found to already harbor Ha-ras mutations, whereas 33% of carcinomas showed various mutations in Ha-ras, Ki-ras, and/or p53. Mutations in Ha-ras may, therefore, be an early event in a significant portion of PIN lesions. Because high-grade PIN showed many characteristics similar to those observed in carcinomas and high-grade PIN was often found contiguous to carcinomas, we conclude that high-grade PIN is a precursor lesion of prostate carcinoma in this transgenic model. These transgenic mice will be useful to study mechanisms responsible for the progression of invasive carcinomas from PIN precursor lesions, as may occur during the development of prostate cancer in humans.

L-ascorbic acid amplification of bladder carcinogenesis promotion by K2CO3.

The dose dependence of K2CO3 promotion of two-stage urinary bladder carcinogenesis and the amplifying effects of additional L-ascorbic acid (AsA) administration were investigated. Male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks and then fed basal diet containing K2CO3 at levels of 0, 1, 1.5, 2.2, and 3% with or without 5% AsA or 3% NaHCO3 supplementation from weeks 5 to 8 (4 weeks) and weeks 12 to 20 (9 weeks). During weeks 9 to 11 (3 weeks), the rats were fed 3% uracil in their diet. For controls, rats without N-butyl-N-(4-hydroxybutyl)nitrosamine treatment were given either 3% K2CO3, 5% AsA, or both plus the uracil treatment. The total observation period was 20 weeks. K2CO3 dose dependently increased the numbers of the putative preneoplastic lesion, papillary or nodular hyperplasia, and papillomas in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine. AsA (5%), while itself exerting no promoting effect, amplified the enhancing influence of K2CO3 on the induction of papillary or nodular hyperplasia and papillomas. The dose-dependent elevation of urinary pH and K+ concentration was associated with K2CO3 treatment with or without AsA. Thus, increased urinary pH and K+ concentration appear to play important roles in K2CO3 promotion, and AsA amplifies this promotion.

Estrogen promotes mammary tumor development in C3(1)/SV40 large T-antigen transgenic mice: paradoxical loss of estrogen receptoralpha expression during tumor progression.

Although several lines of epidemiological evidence suggest that estrogen exposure influences the incidence of breast cancer development, the mechanisms by which estrogen may stimulate the formation of breast cancer remain poorly understood. We have explored how alterations in estrogen exposure can influence the development of mammary cancer in the C3(1)/T(AG) transgenic model, where estrogen levels and estrogen receptor alpha (ERalpha) expression do not appear to modify the level of transgene expression. The C3(1)/T(AG) transgene becomes transcriptionally active in mammary ductal target cells at 3 weeks of age after the estrogen-induced differentiation of the mammary epithelial anlage to the ductal outgrowth stage. Complete maturation of the mammary ductal tree, however, is not required for cancer development because tumors arise in animals where ductal branching and terminal end bud formation have been prematurely arrested by ovariectomy. Mammary tumorigenesis in this model is promoted by increased estrogen exposure with the development of significantly more mammary intraepithelial neoplastic lesions and carcinomas associated with accelerated malignant conversion. The promotion of mammary tumors in this model appears to occur through an estrogen-induced proliferation and increase in the number of available target cells for transformation at the terminal ductal lobular units, as has been postulated to occur in women who receive hormone replacement therapy and/or by additional molecular mechanisms. We show, for the first time in a transgenic mouse model, that mammary tumor progression is associated with the loss of ERalpha expression, as has been often observed in human breast cancers with important clinical significance. Estrogen signaling may, therefore, serve different functions, depending upon the stage of tumorigenesis. ERbeta expression is up-regulated during tumor progression, although the functional significance of this remains to be determined.

L-ascorbic acid amplification of second-stage bladder carcinogenesis promotion by NaHCO3.

The dose dependence of NaHCO3 promotion of urinary bladder carcinogenesis and the effects of additional L-ascorbic acid (AsA) administration were investigated subsequent to initiation. Male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks and then, starting 3 days after cessation of carcinogen treatment, received basal diet containing NaHCO3 at levels of 0, 0.375, 0.75, 1.5, and 3.0% with or without a 5% AsA supplement for 32 weeks. NaHCO3 dose-dependently increased the incidence and numbers of urinary bladder carcinomas in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine. 5% AsA, while itself exerting no promoting effect, amplified the enhancing influence of NaHCO3 on induction of urinary bladder carcinomas. The same dose-dependent elevation of urinary pH and Na+ concentration was associated with NaHCO3 treatment with or without AsA. NaHCO3 significantly increased DNA synthesis in the urinary bladder epithelium and the additional treatment with AsA was associated with a significant further elevation. Thus, increased urinary pH and Na+ concentrations appear to play important roles in NaHCO3 promotion and AsA amplified this promotion. NaHCO3 treatment, with or without AsA, induced cellular proliferation, although it is unclear whether this is an essential factor.

2-difluoromethylornithine and dehydroepiandrosterone inhibit mammary tumor progression but not mammary or prostate tumor initiation in C3(1)/SV40 T/t-antigen transgenic mice.

Female transgenic mice that express SV40 T/t antigens under the regulatory control of the rat C3(1) gene spontaneously develop multifocal mammary lesions that predictably evolve into invasive, hormone-independent carcinomas, whereas male mice are prone to develop prostate cancer. Chemopreventive agents were administered to female C3(1)/SV40 large T-antigen mice from 7 to 19 weeks of age, during which time the mammary lesions developed and progressed to invasive carcinomas. No significant differences in the numbers of preinvasive mammary intraepithelial neoplasia lesions (histologically similar to human ductal carcinoma in situ) were observed after 2 or 8 weeks of treatment between mice receiving either vehicle alone, dehydroepiandrosterone (DHEA), or 2-difluoromethylornithine (DFMO). However, a dose-response reduction in invasive carcinoma growth was observed for both DFMO, an inhibitor of ornithine decarboxylase, and DHEA, the primary steroid precursor to both androgens and estrogens in primates. Despite unaltered expression of the transgene, tumor incidence was reduced approximately 20% by DFMO (8000 mg/kg) and 30% by DHEA (4000 mg/kg; P < 0.05). Tumor multiplicity was reduced by approximately 50% by both DFMO and DHEA (P < 0.05). DFMO had a dose-dependent effect on total tumor burden, which was reduced by 25% at low doses (4000 mg/kg) and 70% at high doses (8000 mg/kg). DHEA reduced tumor burden by 50% and 66% at low (2000 mg/kg) and high (4000 mg/kg) doses, respectively. Interestingly, despite its inhibitory effects on tumor development, DHEA caused a dose-dependent increase of serum estradiol levels that we have previously shown to increase mammary tumor formation in this model. No effect on the development of the prostate cancer precursor lesions (prostate intraepithelial neoplasia) was observed when mice were treated with DHEA, DFMO, tocopherol acetate, selenomethionine, or 9-cis-retinoic acid, although the effects on late-stage prostate cancer development were not determined. These results demonstrate that despite the expression of the highly transforming C3(1)/SV40 large T-antigen transgene, this transgenic model can be used to study the effects of chemopreventive agents on mammary cancer progression. The tumor-inhibitory effects of DHEA and DFMO on mammary cancer growth appear to occur after the development of preinvasive lesions, suggesting that these agents inhibit tumor progression but not initiation.

Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens.

Arsenic (As) is environmentally ubiquitous and an epidemiologically significant chemical related to certain human cancers. Dimethylarsinic acid (cacodylic acid; DMA) is one of the major methylated metabolites of ingested arsenicals in most mammals. To evaluate the effects of DMA on chemical carcinogenesis, we conducted a multiorgan bioassay in rats given various doses of DMA. One-hundred twenty-four male F344/DuCrj rats were divided randomly into 7 groups (20 rats each for groups 1-5; 12 rats each for groups 6 and 7). To initiate multiple organs and tissues, animals in groups 1-5 were treated sequentially with diethylnitrosamine (100 mg/kg body weight, i.p., single dose at the commencement) and N-methyl-N-nitrosourea (20 mg/kg body weight, i.p., 4 times, on days 5, 8, 11, and 14). Thereafter, rats received 1,2-dimethylhydrazine (40 mg/kg body weight, s.c., 4 times, on days 18, 22, 26, and 30). During the same period, the animals were sequentially administered N-butyl-N-(4-hydroxybutyl)nitrosamine (0.05% in the drinking water, during weeks 1 and 2) and N-bis(2-hydroxypropyl)nitrosamine (0.1% in the drinking water, during weeks 3 and 4; DMBDD treatment). After a 2-week interval, groups 2-5 were given 50, 100, 200, or 400 ppm DMA, respectively, in the drinking water. Groups 6 and 7, which were not given DMBDD treatment, received 100 and 400 ppm DMA during weeks 6-30. All rats were killed at the end of week 30. In the initiated groups (groups 1-5), DMA significantly enhanced the tumor induction in the urinary bladder, kidney, liver, and thyroid gland, with respective incidences in group 5 (400 ppm DMA) being 80, 65, 65, and 45%. Induction of preneoplastic lesions (glutathione S-transferase placental form-positive foci in the liver and atypical tubules in the kidney) was also significantly increased in DMA-treated groups. Ornithine decarboxylase activity in the kidneys of rats treated with 100 ppm DMA was significantly increased compared with control values (P < 0.001). In conclusion, DMA is acting as a promoter of urinary bladder, kidney, liver, and thyroid gland carcinogenesis in rats, and we speculate that this may be related to cancer induction by As in humans.

p53-independent apoptosis during mammary tumor progression in C3(1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from preneoplasia to carcinoma.

Alterations in apoptosis and associated mechanisms during mammary tumor progression were investigated in transgenic mice expressing the SV40 large T antigen (T(AG)) driven by the rat prostatic steroid-binding protein C3(1) 5'-flanking region. Apoptosis levels, assessed by in situ end labeling, were low in normal mammary epithelial cells, highest in atypical hyperplasias (preneoplastic lesions), and less pronounced in adenocarcinomas. Preneoplastic cells maintain the ability to undergo apoptosis as a mechanism of tumor growth suppression, but this critical control of apoptosis is lost as these lesions progress to carcinomas. These alterations in apoptosis occur during mammary tumor progression in mice containing wild-type p53+/+ genotype as well as in mice with the p53-/- genotype. Thus, apoptosis in this tumor model occurs through a p53-independent mechanism. Because other studies have demonstrated p53-dependent apoptosis in T(AG)-induced choroid plexus tumors of transgenic mice, we propose that the role of p53 in apoptosis may be tissue-specific. In addition, bcl-2 protein was not expressed in any mammary lesions. SV40 T(AG) expression, which correlated with the nuclear p53 protein at all stages of tumor progression, was low in normal mammary epithelial cells, moderately high in atypical hyperplasias, and strongly expressed in adenocarcinomas. No p53 mutations were found at any stage of mammary adenocarcinoma development, suggesting that tumor progression does not require a dominantly acting p53 mutation in this transgenic model. p2l(Waf1/Cip1), a cyclin-dependent kinase inhibitor, was expressed in normal mammary tissue but was not detected in the mammary carcinomas, despite high nuclear accumulation of wild-type p53 protein, suggesting functional loss of p53 due to binding of SV40 T(AG), to p53. These findings suggest that suppression of apoptosis during the transition from atypical hyperplasia to adenocarcinoma appears to be a critical event for mammary cancer development in C3(1)/T(AG) transgenic mice and occurs by p53- and bcl-2-independent pathways.

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice.

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5'-flanking region display multistage mammary tumorigenesis. To delineate genetic changes associated with mammary tumor progression, comparative genomic hybridization (CGH) was performed. CGH revealed a consistent gain of the telomeric region of chromosome 6. This region contains the Ki-ras proto-oncogene. Analyses of genomic DNA by Southern blot demonstrated up to 40-fold amplification of the Ki-ras gene. Ki-ras amplification was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no amplifications were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene amplification exhibited high levels of Ki-ras RNA and protein. The over-expressed Ki-Ras protein in these tumors appeared functionally active as indicated by the elevated MAP kinase activity. These data demonstrate that while Ki-ras amplification might not be an early event, there is a strong association between Ki-ras amplification and over-expression and mammary tumor progression in this model. This study also shows that CGH is a powerful and useful technique for identifying chromosomal copy number changes during tumor progression, and that this model may provide a predictable in vivo system for studying gene amplification.

Haploid loss of Ki-ras delays mammary tumor progression in C3 (1)/SV40 Tag transgenic mice.

We have previously demonstrated that amplification and overexpression of the Ki-ras gene is associated with mammary tumor progression in C3(1)/SV40Tag transgenic mice (Liu et al., 1998). To further evaluate the functional significance of the Ki-ras proto-oncogene in mammary cancer development, in vivo studies were conducted to examine the effect of Ki-ras gene dosage on tumor progression. The lack of one normal Ki-ras allele C3(1)/SV40Tag transgenic mice resulted in significantly delayed mammary intraepithelial neoplasia (MIN) formation as well as in a decreased number of mammary gland carcinomas. However, despite the retardation of tumor development by reduced Ki-ras gene dosage, overall survival was only modestly affected. This appears to be due to several factors including significant mammary tumor growth associated with Ki-ras gene amplification and over-expression that occurs during the advanced stage of oncogenesis in mice carrying either one or two normal Ki-ras alleles. The retardation of tumor progression due to the haploid loss of Ki-ras did not appear to be related to accelerated apoptosis, or a reduced rate of cell proliferation at the tumor stages examined. These data strongly suggest that the gene dosage of Ki-ras affects tumor promotion at an early stage of mammary tumor progression in this SV40 Tag-induced model of mammary oncogenesis.

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.

The 5' flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-alpha expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

Heterotopic endochondrial ossification with mixed tumor formation in C3(1)/Tag transgenic mice is associated with elevated TGF-beta1 and BMP-2 expression.

Transgenic mice which express the simian virus 40 large T-antigen (Tag) under the regulatory control of the hormone responsive rat C3(1) gene develop unusual lesions of heterotopic bone growth associated with mixed tumor formation arising from eccrine sweat glands found only in the foot pads of mice, ischiocavernosus muscle adjacent to bulbourethral glands and occasionally the salivary and mammary glands. These lesions are very similar to mixed tumors arising in several types of human cancers. Based upon electron microscopic examination and immunocytochemical analyses of cellular differentiation markers, the mixed proliferative lesions in this transgenic mouse model begin with the Tag-induced proliferation of epithelial and myoepithelial cells. The proliferation of these two types of cells results in hyperplasia and adenomatous transformation of the epithelial component, whereas the proliferating myoepithelial cells undergo metaplasia to form chondrocytes which deposit extracellular matrix, including collagen fibers. Cartilage develops focally between areas of epithelial proliferation and subsequently ossifies through a process of endochondrial bone formation. The metaplasia of myoepithelial cells to chondrocytes appears to require the inductive interaction of factors produced by the closely associated proliferating epithelial cells, including members of the TGF-beta superfamily. We demonstrate that TGF-beta1 protein accumulates in the extracellular matrix of the lesions, whereas RNA in situ hybridization reveals that BMP-2, another strong inducer of heterotopic bone formation, is overexpressed by the proliferating epithelial cells during the development of ectopic bone. The formation of sarcomatous tumors within the mixed tumors appears to be androgen-dependent and more frequent in mice lacking a normal allele of p53. This process of cartilage and bone induction may mimic epithelial-mesenchymal interactions which occur during embryonic bone formation. These transgenic mice may provide new insights into the processes of ectopic endochondrial bone formation associated with mixed tumor formation and serve as a useful model for human heterotopic bone disease.

Suppression of mammary carcinoma growth in vitro and in vivo by inducible expression of the Cdk inhibitor p21.

Mammary carcinomas that develop in C3 (1)/SV40 T- antigen (TAg) transgenic mice have lost the p53-mediated induction of p21, leading to increased cellular proliferation and significant elevations of cyclins and Cdks. To test whether p21 could serve as a target for anticancer therapy for this mammary cancer model, a retroviral delivery system for the inducible expression of p21 was developed. We demonstrate that overexpression of p21 in C3(1)/TAg mammary tumor cells using the retroviral inducible p21 expression system results in increased apoptosis, reduced cell proliferation in vitro and reduced tumor growth in vivo associated with reduced expression of cyclins D1 and E, and Cdks 2, 4, and 6. Reciprocal changes in the expression of p21 and p27(Kip1), another cell-cycle regulator, were also observed. Because reduced p21 expression occurs frequently in human breast cancer, restoration of the Cdk inhibitor p21 by gene therapy approaches may provide a method for inhibiting mammary tumor progression.

Proliferative response of renal pelvic epithelium in rats to oral administration of ortho-phenylphenol, sodium ortho-phenylphenate and diphenyl.

Changes in DNA synthesis levels and morphology as observed by scanning electron microscopy (SEM) and light microscopy in rat renal papilla and pelvis following oral administration of ortho-phenylphenol (OPP), sodium ortho-phenylphenate (OPP-Na) and diphenyl, were investigated in F344 rats. OPP and OPP-Na treatment for 4 weeks was associated with elevated DNA synthesis in both renal papilla and pelvis, in addition to distinct morphological cell surface alterations. Sequential light microscope observation revealed induction of renal papillary necrosis from week 4, followed by regeneration hyperplasia at weeks 16 and 24, but no changes in the renal pelvis in the OPP case. OPP-Na not only similarly affected the renal papilla, but also brought about development of hyperplasia in the renal pelvis. No proliferative response of the kidney was apparent in rats fed diphenyl. The present study indicated that the proliferative responses in the renal pelvic epithelium following OPP-Na are similar to those induced by this chemical in the urinary bladder, and that in both cases they are indicative of promoting or carcinogenic potential.

Absence of promotion potential for calcium L-ascorbate, L-ascorbic dipalmitate, L-ascorbic stearate and erythorbic acid on rat urinary bladder carcinogenesis.

The effects of treatment with calcium L-ascorbate, L-ascorbic dipalmitate, L-ascorbic stearate and erythorbic acid on two-stage urinary bladder carcinogenesis in F344 rats after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Carcinogen was administered at a dose of 0.05% in drinking water for 4 weeks and thereafter the test chemicals were given as a 5% supplement in the diet for the following 32 weeks. No increase in the induction of preneoplastic lesions, papillomas or carcinomas was apparent and it was concluded that none of the test chemicals possess promoting activity for urinary bladder carcinogenesis.

Lack of involvement of p53 gene mutations in N-methyl-N-nitrosourea-induced bladder tumor progression in N-butyl-N-(4-hydroxybutyl)nitrosamine-treated rats and no suppression by indomethacin.

The relevance of p53 mutations to rat bladder cancer progression induced by a single injection of N-methyl-N-nitrosourea (MNU) and the chemopreventive effects of indomethacin (IM) were investigated in male F344 rats, initially given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at a dose of 500 ppm in the drinking water for 10 weeks. The animals were subsequently treated with a single intraperitoneal injection of MNU at a dose of 50 mg/kg b.w. at week 20. A subgroup was then given IM dissolved in the drinking water at a concentration of 20 ppm for 20 weeks. The experiment was terminated at week 40 when transitional cell carcinomas (TCC) were observed in all animals given BBN, regardless of the administration of MNU and/or IM (incidences ranged from 80 to 100%). The extent of invasion was significantly greater with the additional MNU treatment but no inhibitory effects of IM were noted. A low frequency of p53 mutations was detected without relation to the extent of tumor invasion. Thus, only two mutations were found, one in a Ta and the other in a T1 carcinoma. The present study thus demonstrated that p53 mutations are not involved in MNU-induced progression in rat urinary bladder cancers, suggesting that they are not critical for malignancy.