Induced Rat Mammary Tumors Research Articles

Assessment of induced rat mammary tumour response to chemotherapy using the apparent diffusion coefficient of tissue water as determined by diffusion-weighted1H-NMR spectroscopy in vivo

Chemosensitivity of N-methyl-N-nitrosourea-induced rat mammary tumours treated with 5-fluorouracil at a dose of 100 mg kg(-1) i.p. was assessed by using diffusion-weighted 1H-MRS to measure the average diffusion coefficient (ADC) of water in the tumour tissue. ADC measurements prior to any therapy correlated positively with necrotic fraction. Tumours with low initial ADC (< 0.95 x 10(9) m2 s(-1)) showed an increase in ADC 7 days after treatment, whereas tumours with a high initial ADC (> 1.2 x 10(9) m2 s(-1)) showed a decrease. All tumours decreased significantly in volume (P < 0.05) 2, 5 and 7 days after treatment. At day 7 post-treatment, tumours with a high pre-treatment ADC started to regrow. The initial ADC value, as well as changes after treatment predict tumour chemosensitivity, which could be clinically relevant.

Assessment of induced rat mammary tumour response to chemotherapy using the apparent diffusion coefficient of tissue water as determined by diffusion-weighted 1H-NMR spectroscopy in vivo

Chemosensitivity of N-methyl-N-nitrosourea-induced rat mammary tumours treated with 5-fluorouracil at a dose of 100 mg kg−1 i.p. was assessed by using diffusion-weighted 1H-MRS to measure the average diffusion coefficient (ADC) of water in the tumour tissue. ADC measurements prior to any therapy correlated positively with necrotic fraction. Tumours with low initial ADC (<0.95×109 m2 s−1) showed an increase in ADC 7 days after treatment, whereas tumours with a high initial ADC (>1.2×109 m2 s−1) showed a decrease. All tumours decreased significantly in volume (P<0.05) 2, 5 and 7 days after treatment. At day 7 post-treatment, tumours with a high pre-treatment ADC started to regrow. The initial ADC value, as well as changes after treatment predict tumour chemosensitivity, which could be clinically relevant.

On the statistical analysis of allelic-loss data.

This paper concerns the statistical analysis of certain binary data arising in molecular studies of cancer. In allelic-loss experiments, tumour cell genomes are analysed at informative molecular marker loci to identify deleted chromosomal regions. The resulting binary data are used to infer properties of putative suppressor genes, genes involved in normal cell cycling. Various factors can complicate this inference, including background loss of heterozygosity, spatial (that is, within chromosome) dependence of the binary responses, non-informativeness of markers, covariates such as protein levels or tumour histology, heterogeneity of cells within tumours, and measurement error. We focus on the first three factors, discussing methods for statistical inference that separate background loss from significant loss. We outline the extension to other inferences, such as comparison questions and the relationship to covariates. Using characteristic features of tumourigenesis, we present a framework for the stochastic modelling of allelic-loss data, and build models within this framework; in particular, we propose a simple model that has chromosome breaks at locations of a Poisson process, and preferential selection cells with inactivated suppressor genes. We illustrate these methods on allelic-loss data from induced rat mammary tumours and human bladder cancers.

Alterations in the Ha‐ras‐1 and the p53 pathway genes in the progression of N‐methyl‐N‐nitrosourea–induced rat mammary tumors

Alterations in the Ha‐ras‐1 and the p53 pathway genes in the progression of N‐methyl‐N‐nitrosourea–induced rat mammary tumors

Alterations in the Ha-ras-1 and thep53 pathway genes in the progression ofN-methyl-N-nitrosourea–induced rat mammary tumors

Well-differentiated mammary carcinomas carrying mutated Ha-ras-1 oncogenes arise frequently in pubescent rats exposed to the direct-acting methylating agent N-methyl-N-nitrosourea (MNU). When these tumors are serially transplanted, they acquire more aggressive phenotypes. To determine the genetic alterations underlying local invasion, hormone independence, and metastasis, we studied alterations in the Ha-ras-1, p53, and mdm2 genes in successive generations of tumors passaged in intact or ovariectomized rats. Although previous studies have shown that selective amplification of the mutant Ha-ras-1 allele correlates strongly with the acquisition of hormone independence, we found that the acquisition of an invasive phenotype did not depend on mutational activation or amplification of Ha-ras-1. Mutations in the p53 gene were rare. Of a total of 120 primary, locally invasive, hormone-independent, and metastatic tumors tested for mutations in exons 4-9 of the p53 gene, only one mutation was detected in the later passages of an invasive tumor line. No gross gene alteration or amplification was seen in mdm2, a negative regulator of p53 transcription. Thus, the p53 gene is an infrequent mutational target, and amplification of the mdm2 gene does not appear to play a role in initiation or progression of rat mammary tumorigenesis.

Decreased Egr‐1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene. Int. J. Cancer 72:102–109, 1997. © 1997 Wiley-Liss Inc.

Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation.

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.

IL-1 gene expression in human mammary tumour xenografts after treatment with hexadecylphosphocholine.

Hexadecylphosphocholine (HPC), an alkylphosphocholine, is active against experimental and clinical breast cancer in viw but the precise mechanisms involved in its anti-tumour activity are not fblly understood. In vitro studies have revealed remarkable anti-tumour activity against HL60, U937, Raji and K562 cell lines [l], three human mammary carcinomas cell lines, MT-1, MT-3, MaTu and a murine colon carcinoma MAC 16 [2]. HPC also induced apoptosis in KB cells in vitro [3]. In vivo studies with human mammary carcinomas in nude mice showed tumour volume was reduced following treatment with HPC compared with classic anti-tumour drugs such as doxorubicin, mitoxantrone, methotrexate and 5-fluorourad [4]. Topical application to recurrent breast cancer in the skin has resulted in a 30?? overall response rate [S]. In addition, HPC had outstanding anti-tumour activity in dimethylbenzanthe (DMBA) induced rat mammary tumours, however, another autochthonus tumour, the benzo (a) pyrene-induced sarcoma of the rat did not respond to HPC treatment 161. HPC has been shown previously to have effects on the immune system in vitro such as amplifying the efFect of granulocyte colony-stimulating factor on haematopoietic cells [7] and in liposomal form, increasing the production of TNFa [S]. Increases in leucocyte and platelet counts in patients after administration of HPC have also been observed [9]. Preliminary studies in this laboratory have shown increased expression of CD3 antigen in spleens [lo] and morphological changes in secondary immune tissues [ 111 and tumours [ 121 of MT-I tumour bearing nude mice after treatment with HPC. As yet, however, there is no clear understanding of how this drug recruits cells of the immune system or if immunological modifications lead to anti-tumour effects. The aim of this study was to investigate the anti-tumour mechanisms of HFT against MT1 breast cancer xenografts growing in nude mice and in particular the role of the immune system including cytokine production in the regression of tumours. MT-I tumours were transplanted into the mammary fat pads of twenty four female twelve week old NCWnude mice (weight range 25-30g) subcutaneously by means of a trocar. Tumours were allowed to grow until they were large enough to be measured accurately by callipers (approximately 22 days). Eight of these mice were then used for treatment with HPC, 8 mice were treated with solvent, 4 mice were treated with cyclophosphamide and 4 were kept as untreated controls. HPC was solubilized in 1 O?? Tween 80/ saline and administered orally once a day for 5 days to MT-1 tumour-bearing nude mice at a daily dose of 5Omg/kg. Both untreated and solvent treated controls were included. Cyclophosphamide was solubilized in saline and administered as a single I.P. dose (25Omg/kg). One week after the end of drug administration, the mice were killed and the tumours removed for histology, immunostaining and RNA extraction. Mice treated with cyclophosphamide were sacrificed when the tumours started to regress. Control mice were also sacrificed at the same time and the tumours removed for similar evaluation. Endothelial cells, macrophages (Mcp) and lymphocytes were detected with antibodies to CD31, pan Mcp marker, CD3, CD4, CDS and CD45wB220 respectively. To investigate the production of cytokines, the expression of IL-l was assessed by RT-PCR. HPC treated tumours showed large areas of lysis and the presence of giant cells. Immunostaining revealed increases in endothelial cells associated with massive infiltration of Mcp, T cells (Th and Tc) and B cells. Immune cell infiltration was not observed in tumours following cyclophosphamide treatment or control tumours. In hrther experiments to investigate cytokine gene expression, IL-l mRNA was detected in HPC treated tumours but not in cyclophosphamide treated or control tumours using RT-PCR (Fig. 1). 1 2 3 4 5 6

The increase of apoptosis and the decrease of proliferation by angiogenesis inhibitor in rat mammary tumors

We have investigated the change of balance between proliferating and apoptotic tumor cells in dimethylebenz(a)-anthracene (DMBA)-induced rat mammary tumors by using anti-angiogenic drug, AGM-1470 and anticancer drug, 5'-deoxy-5-fluorouridine (5'-DFUR). The rate of PCNA positive proliferating cells was decreased in the tretment groups, especially in AGM-1470 and in the combination of AGM-1470 and 5'-DFUR. And apoptotic index evaluated by TUNEL method was increased in 5'-DFUR and in the combination group. The ratio of proliferating cells/apoptotic cells was markedly decreased in AGM-1470 and AGM+5'-DFUR group compared to control group. These data suggested that anti-angiogenic drugs might regulate the growth behavior of tumor cells by changing the microenvironments.

Protective effect of leuprolide acetate on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats

The anti-carcinogenic effect of TAP-144-SR biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent LHRH agonist, TAP-144 (D-Leu6-(des-Gly10-NH2)-LHRH ethylamide, leuprolide acetate) was investigated in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumours. At 50 days of age, all rats were given 20 mg doses of DMBA by gastric intubation. Rats on the Initiation Group were given a single s.c. injection of TAP-144-SR (3 mg/kg) 1 week before DMBA treatment, and rats in the Promotion Group were given repeated s.c. injections every fourth week from 1 week after DMBA treatment until the end of the experiment. In the Initiation Group, tumour incidence and multiplicity were reduced to 20.8% and 15.0% of the Control Group values, respectively. In the Promotion Group, tumour incidence and multiplicity were also reduced to 17.7% and 7.9%, respectively. It was suggested that TAP-144-SR may be a potential candidate as an agent for hormonal chemoprevention in breast cancer.

Incidence of p53 and Ha-ras gene mutations in chemically induced rat mammary carcinomas.

To determine whether p53 alterations, which are frequent in human breast cancers, are also common in rat mammary tumors, we examined 40 tumors from 24 rats treated with 7,12-dimethylbenz[a]anthracene (DMBA) and 34 tumors from 14 rats treated with N-nitroso-N-methylurea (NMU) (an N-nitroso compound). DMBA and NMU are known genotoxic mutagens. The entire coding regions of the p53 and Ha-ras genes were examined for mutations by polymerase chain reaction single-strand conformational polymorphism analysis and by direct sequencing. One of the 40 DMBA-induced mammary tumors had a p53 mutation, a single-base substitution (AGC-->GGC) at codon 307, resulting in an amino-acid change from Ser to Gly. No mutations were found in NMU-induced tumors. The incidence of Ha-ras gene mutation was 79% (27 of 34) at codon 12 in the NMU group and 23% (nine of 40) at codon 61 in the DMBA group. Thus, p53 mutation, in contrast to Ha-ras mutation, did not seem to be a prerequisite for carcinogenesis in chemically induced rat mammary tumors.

Evidence of in situ estrogen synthesis in nitrosomethylurea-induced rat mammary tumors via the enzyme estrone sulfatase.

A variety of indirect evidence suggests that mammary tumors can synthesize free estrogens in situ via the sulfatase enzyme. The present study utilized an isotopic kinetic technique to provide direct confirmation of local tumor synthesis. Animals bearing nitrosomethylurea (NMU)-induced rat mammary tumors were infused with 14C-estrone as well as 3H-estrone sulfate and plasma:tissue gradients for each steroid measured. Liver, serving as a control tissue, uniformly synthesized free estrone from estrone sulfate with local synthesis in this organ providing an average of 78 +/- 1.0% of the estrone in this tissue. In rat mammary tumors, five out of seven synthesized estrone locally with individual values ranging from 19 to 50% synthesized in tissue. These data indicate that liver uniformly converts estrone sulfate to free estrone, whereas the majority, but not all, breast tumors synthesize estrogen locally via this pathway.

Usefulness of 22-oxa-1,25-dihydroxyvitamin D-3 (OCT) as a single agent or combined therapy with aromatase inhibitor (CGS 16949A) on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors

The antitumor effects of 22-oxa-1,25-dihydroxy-vitamin D-3 (OCT), a vitamin D-3 analogue, were evaluated on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumors. The combined effects of OCT (0.3 mu g/kg) with tamoxifen (0.5 mg/kg) medroxyprogesterone acetate (MPA) 2.5 mg/kg), or a new aromatase inhibitor, CGS 16949A (0.8 mg/kg) were also evaluated. OCT significantly suppressed the growth of tumors without hypercalcemia in a dose dependent manner at the fourth week from the start of treatment. Tumor size in the OCT+CGS 16949A group was significantly decreased compared with that in the OCT or CGS 16949A alone. However, there was no significant difference in tumor size between OCT alone and combined therapy with tamoxifen or MPA. We conclude that a single administration of OCT, which does not cause hypercalcemia, is effective for breast cancer and that a combination of OCT and aromatase inhibitor, CGS 16949A augments the antitumor effect on tumors compared to each single agent.

Brief communication, P53 accumulation in N-methyl-N-nitrosourea-induced rat mammary tumors.

P53 aberrant protein expression and mutation is a component of many human tumors but less information is available regarding involvement in relevant animal models. We have examined invasive mammary epithelial neoplasms for p53 aberrant protein expression from Sprague-Dawley rats induced by two intrajugular injections of N-methyl-N-nitrosourea (NMU, 50 mg/kg), 7 days apart, beginning at 44-49 days of age. Positive nuclear staining was observed in 8/10 mammary tumor frozen sections using PAb 421, a mouse monoclonal antibody, compared to a mixed mouse IgG negative control antibody. Paraffin sections from two additional sets of similarly induced rat mammary tumors also showed positive nuclear staining (22/37 tumors) with CM-5, a rabbit polyclonal antibody. Our results indicate that elevated cellular content of p53 is a common event in invasive palpable mammary tumors induced by NMU in this dual-injection model system.

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea.

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.

Ha-ras-1 oncogene mutations in mammary epithelial cells do not contribute to initiation of spontaneous mammary tumorigenesis in rats.

We have previously shown that oncogenic GGA to GAA mutations in codon 12 of the Ha-ras-1 gene arise spontaneously during normal development of the mammary epithelium of female Fischer 344 (F344) rats. Our result further demonstrated that the vast majority of nitrosomethylurea (NMU)-induced rat mammary tumors with Ha-ras-1 oncogenes arose from these pre-existing mutants. We therefore investigated whether Ha-ras-1 mutants acquired a selective growth advantage in vivo in the absence of NMU exposure. Our results indicated that between the ages of 50 and 570 days, the total number mammary epithelial cells per rat increased approximately 5 fold (from 3.7x10(7) to 1.8x10(8) cells), while the average number of Ha-ras-1 mutants per rat increased approximately 25 fold (from 160 to 4000 cells). Thus, the mutants acquired a measurable (5-fold) growth advantage in vivo. To determine if the growth of these mutants contributed to spontaneous mammary carcinogenesis, we measured Ha-ras-1 mutant fractions in 26 tumors from untreated F344 rats. The assay failed to detect Ha-ras-1 mutant fractions higher than 10(-3), indicating that in the mammary epithelium, the activating mutation of the Ha-ras-1 gene is a conditional oncomutation, whose oncogenic potential is realized only under certain specific physiological conditions, such as exposure to a carcinogenic dose of NMU exposure.

COMBINATION EFFECT OF AN ANGIOGENESIS INHIBITOR AGM-1470 WITH 5'-DEOXY-5-FLUOROURIDINE, AND WITH HORMONAL DRUGS IN DMBA-INDUCED RAT MAMMARY-TUMORS

AGM-1470, a potent angiogenesis inhibitor, is known to suppress the growth of solid tumors in vivo. In this study, we investigated the combination effect of AGM-1470 with 5'-deoxy-5-fluorouridine (5'-DFUR) which is converted to 5-FU by pyrimidine nucleoside phosphorylase, with tamoxifen (TAM), and with medroxyprogesterone acetate (MPA) in dimethylebenz(a)-anthracene (DMBA) induced rat mammary tumors. Since 5'-DFUR and MPA are noted to have antiangiogenic activity, this combination study might imply not only the addition to chemotherapeutic or hormonal drugs for the angiogenesis inhibitor but also the combination of two angiogenesis inhibitors. The combination of AGM-1470 (20 mg/kg) with 5'-DFUR (200 mg/kg) and with TAM (0.8 mg/kg) markedly suppressed the tumor growth. There was a significant difference in the suppression of tumor growth between the combination group and either drug alone group (p<0.01). No statistical combination effect between AGM-1470 and MPA was found, however MPA markedly reversed the body weight loss induced by AGM-1470. This result will be informative for clinical studies on the combination effect of AGM-1470.

Growth inhibition of DMBA-induced rat mammary carcinomas by the antiandrogen flutamide.

Antiandrogens have sporadically been reported to exert antitumor activities in both pre- and post-menopausal breast cancer. To explore the possibility of using the pure antiandrogen flutamide (FLU) in breast cancer therapy, rats bearing DMBA-induced mammary tumors were treated with FLU, dihydrotestosterone (DHT), or FLU plus DHT. FLU was administered orally, at doses comparable to those used in the treatment of prostate cancer patients. FLU-treated animals had a significantly smaller average tumor area than controls from day 11 up to the end of the experiment (day 20). A similar reduction of tumor growth was observed in rats given DHT and in those treated with DHT plus FLU. Plasma levels of LH, FSH, P, 17-OH P, E2 and DHEA measured at the end of experiment did not differ between treated animals and controls. Results demonstrate that the antiandrogen FLU and the full androgen DHT exert similar inhibitory effects on the growth of dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors. Moreover, data show that plasma steroids levels are unaffected by FLU treatment. This finding rules out any antitumor effect dependent on the reduction of adrenal and gonadal steroidosynthesis, and makes it appear more likely that androgen receptors are involved in the antiproliferative effect of FLU.

Cell Loss in Dimethylbenz(a)anthracene&amp;ndash;lnduced Rat Mammary Carcinoma Treated with Toremif ene and Ovariectomy

The antiestrogen toremifene treatment results in regression or stabilization of dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors by reducing the mitotic activity and possibly by modifying the abundant spontaneous apoptosis seen in these tumors. A comparative study on the modes of cell loss was performed in untreated DMBA tumors and in tumors treated with toremifene and ovariectomy. Ovariectomy results in massive apoptotic cell death throughout the tumors with apoptotic cells exfoliating into the glandular lumina. Apoptosis accounts for most of the cell death also in the untreated tumors. The distinctly different morphology of the apoptotic cells in the untreated tumors (condensed apoptosis) and tumors treated with ovariectomy (blebbing apoptosis) may reflect the different type or schedule of the apoptotic process. Both morphologies of apoptotic cells were seen in the toremifene-treated tumors. Blebbing apoptosis is not easily examined in paraffin-embedded material but is best detectable in plastic sections and by electron microscopy.

Analysis of caspase activities in rat mammary tumours induced by N-methyl-nitrosourea

Normal breast development is controlled by a balance between cell proliferation and apoptosis. The balance between the two parameters is crucial for determining the growth or regression of breast tumours in response to therapies and treatments. Therefore, it is necessary to understand the role of apoptosis in tumour progression. Active caspases participate as essential elements in the execution of apoptotic mechanisms. In the present study, we analysed the activities of caspase-3, -8 and -9 as well as cytochrome c release in N-methyl-nitrosourea (NMU)-induced rat mammary tumours, in order to establish the apoptotic events that occur in tumour growth in this animal model. Forty female virgin Wistar rats were randomly divided into two groups. One group was injected intraperitoneally with three doses of 50 mg/kg body weight of NMU. The control group received the vehicle only. After 122 days of NMU injection, the rats were sacrificed and the tumours were excised and processed. Results showed that in mammary tumours induced by NMU, the apoptotic death receptor-mediated pathway is activated through caspase-3 and -8, but the apoptotic mitochondrial pathway is suppressed through a non-activating process of caspase-9 activity, despite the release of cytochrome c. In conclusion, these findings have demonstrated a suppression of the apoptotic mitochondrial pathway through a non-activating process of caspase-9 activity, despite the release of cytochrome c in mammary tumours induced by NMU. Although the apoptotic death receptor-mediated pathway is activated, it is not enough to maintain the balance between proliferation and apoptosis, and thus determine the overall growth of the tumour.