Absence of Ras mutations in rat DMBA-induced mammary tumors

Animal cancer models reduce genetic background heterogeneity and thus, may facilitate identification and analysis of specific genetic aberrations in tumor cells. Rat and human mammary glands have high similarity in physiology and show comparable hormone responsiveness. Thus, spontaneous and carcinogen (e.g., NMU and DMBA)-induced rat mammary models are valuable tools for genetic studies of breast cancer. In NMU-induced rat mammary tumors, activating mutations in Hras codon 12 have frequently been reported and are supposed to contribute to the mammary carcinogenic process. Involvement of Ras mutations in DMBA-induced tumors is less clear. In the present study we investigated the mutation status of the three Ras genes, Hras, Kras, and Nras, in DMBA-induced rat mammary tumors. We examined codons 12, 13, and 61 of all three genes for mutations in 71 tumors using direct sequencing method that in experimental conditions is sensitive enough to detect single nucleotide mutations even when present in only 25% of the test sample. No activating Ras gene mutation was found. Thus, in contrast to NMU-induced rat mammary tumor, tumorigenesis in DMBA-induced rat mammary tumors seems to be independent on activating mutations in the Ras genes. Our finding suggests that the genetic pathways selected in mammary tumor development are influenced by and perhaps dependent on the identity of the inducing agent, again emphasizing the importance of tumor etiology on the genetic changes in the tumor cells.     

Adrenal steroids stimulate growth and progesterone receptor levels in rat uterus and DMBA-induced mammary tumors

We have studied the effect of treatment with the adrenal steroids androst-5-ene-3,17-diol (⁵-diol) and dehydroepiandrosterone (DHEA) on the growth and progesterone receptor levels of dimethylbenz-(a)anthracene (DMBA)-induced mammary tumors in the rat.While the total number of tumors in ovariectomized animals was 0.60 ± 0.19 tumor per rat after 24 days, it increased to 2.54 ± 0.50 (p<0.01) and 1.42 ± 0.26 (p<0.01) in the ⁵-diol and DHEA (2 mg, twice daily) treated animals, respectively. While very few new tumors developed during a 24-day period in ovariectomized animals (0.07 ± 0.07/rat), an average of 0.47 ± 0.19 (p<0.05) new tumor per animal appeared in intact rats. In ovariectomized animals treated with ⁵-diol or DHEA, the numbers of new tumors were 0.77 ± 0.26 (p<0.05) and 0.42 ± 0.15 (p<0.05), respectively. An even more striking effect was observed on average total tumor area, which decreased from 4.70 ± 0.95 cm² in intact animals to 0.75 ± 0.27 cm² (p<0.01) following ovariectomy. Values of 9.79 ± 2.25 (p<0.01) and 3.93 ± 0.86 cm² (p<0.01) were found in the ⁵-diol- and DHEA-treated ovariectomized animals, respectively.Treatment of ovariectomized animals with ⁵-diol and DHEA caused a marked increase (p<0.01) in progesterone receptor levels in both the uteri and DMBA-induced mammary tumors. Uterine weight was also stimulated (p<0.01) by treatment with the two adrenal steroids.The present data show that two adrenal C₁₉-⁵ steroids, ⁵-diol and DHEA, possess stimulatory effects analogous to those of estrogens on DMBA-induced mammary tumor growth and progesterone receptor levels in the rat, thus supporting the suggestion of an important role of these adrenal steroids in breast cancer and other estrogen-sensitive diseases in the human.     

Hyperoxia retards growth and induces apoptosis,changes in vascular density and gene expression in transplanted gliomas in nude rats

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO₂ = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO₂ = 2.0), whereas animals housed under normal atmosphere (1 bar, pO₂ = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a ∼60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large “empty spaces” within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells (∼21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11–24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses. Increased pO₂−levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.     

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

Summary In order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent -adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize -adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthraene (DMBA) administration in the rat. The binding of [¹²⁵I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [¹²⁵I]CYP binds to a single class of high affinity sites (114 ± 2.1 fmoles/mg protein) at an apparent K_D value of 38.0 ± 0.3 pM. The order of potency of a series of agonists to compete for [¹²⁵I]CYP binding is consistent with interaction with a ₂-subtype receptor: zinterol > (–)isoproterenol > (–)epinephrine» (–)norepinephrine. In addition, the potency of a series of specific ₁, and ₂ synthetic compounds to displace [¹²⁵I]CYP in mammary tumors is similar to their potency in typical ₂-adrenergic tissues. The binding of [¹²⁵I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (–)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [¹²⁵I]CYP performed on frozen sections revealed the presence of specific -adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18–22 months) female rats have high levels of -adrenergic receptors. Castration decreased the concentration of [¹²⁵I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and -adrenergic receptor concentration. The presence of -adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.     

Comparison of nuclear proteins from DMBA-induced mammary tumors and lactating mammary glands by polyacrylamide gel electrophoresis

Summary Nuclear proteins were extracted from purified nuclei of 7,12-dimethylbenz(a)anthracene (DMBA)-induced tumors and normal mammary glands of the rat by enzymatic treatment. Of the 34 bands indicated by onedimensional polyacrylamide gel electrophoresis of nuclear proteins, 6 appeared in high concentration in tumors but were found as traces or undetectable in normal glands; 6 others were clearly shown in the latter but were not detectable or greatly reduced in the former. Two-dimensional electrophoresis identified about 130 and 92 non-histone proteins in normal mammary and tumor cell nuclei respectively. Marked differences in spot density were noted especially in spots (M.W. × 10^–3/pI) 100/5.7 and 200/5.5 of tumors and 28/7.1, 32/5.4, 36/5.4, 38/6.9, and 68/6.0 of normal tissue. The relationship between these nuclear proteins and the development of mammary tumors is also discussed.Supported in part by a grant from the Cancer Research Society Inc., Montreal.     

Hyperbaric oxygen alone or combined with 5-FU attenuates growth of DMBA-induced rat mammary tumors

We tested the hypothesis that hyperbaric oxygen (HBO) alone and with chemotherapy (5-FU) attenuates tumor growth of DMBA-induced tumors in rats. Six series were performed: (1) Controls (air and vehicle 0.9% NaCl i.p.), (2) 5-FU (0.2 mg/kg i.p.), (3) HBO (2 bar for 90 min and vehicle), (4) HBO and 5-FU, (5) HBO (11 days) and air (next 12 days), (6) HBO (23 days). All treatments were applied on days 1, 4, 7, 10 (Series 1-4), as well as on days 14, 17 and 23 (Series 5-6). Tumor diameter increased by 76.7 and 41.2% in untreated controls and in the 5-FU group, respectively, after 10 days. Tumor size fell by 17-24.2% in the HBO groups and by 35.5% when combined with 5-FU (P < 0.05 compared to HBO). HBO treatment reduced the total number of blood vessels in the tumors. After completion of HBO treatment tumor size increased, but statistically insignificant, during the next 12 days.     

Biological significance of interstitial collagenase in DMBA-induced mammary tumors of the rat

Summary In this review the production of interstitial collagenase in DMBA-induced mammary tumors of the rat has been examined. Cell sorting and cell cultures have given us the opportunity to relate the release of collagenase to a specific cell type. By means of FITC-fluorescence and monospecific antibodies (S. Sakamoto, Harvard University, Boston) it was further possible to localize collagenase in vitro and in vivo. The most outstanding characteristic is that collagenase is produced both by cuboidal, epithelial cell and by macrophages in vitro but not by myoepithelial-like cells. On the other hand, synthesis of collagenase in vivo was detected in some stromal cells, possibly macrophages, but not in neoplastic cuboidal cells. This observation has been related to the inability of cuboidal cells to interact with stromal, fibrillar collagen in vivo since tumor cells are arranged in glandular-like structures bordered by myoepithelial cells and a basement membrane.In vitro, fibrillar rat tail tendon collagen was found to be a potent stimulator of collagenase production by cuboidal cells. Collagenase stimulation by interstitial collagen therefore suggests a plausible mechanism for the degradation of collagen fibrils during local invasion by mammary tumor cells.Part of this work was supported by the National Cancer Institute Bethesda, USA (Contract NO1-CB-74109).     

Intrinsic differences in the transplantability and outgrowth potential of DMBA-induced rat mammary tumors

Only a small proportion of DMBA-induced rat mammary tumors are transplantable in cleared (gland-free) mammary fat pads. To enhance their transplantability, several host factors were modified: age, parity, hormone levels, and tissue integrity of the fat pad. Our 2 important findings were that (1) there were individual differences between the tumors in their transplantability and potential to proliferate normal and hyperplastic outgrowths, and (2) these differences were determined by their inherent potential and not by their environment. These intrinsic differences among the tumors are consistent with the concept that properties of a given type of tumor are independently variable.     

Expression of the c-Ha-ras and neu oncogenes in DMBA-induced, anti-estrogen-treated rat mammary tumors

Dimethylbenzanthracene (DMBA)-induced rat mammary tumors were analyzed for the structure and expression of the oncogenes c-Ha-ras and neu and the effects of anti-estrogen treatment. Tumor samples were divided into 3 groups, the first consisting of untreated tumors, the second of anti-estrogen (toremifene)-treated unresponsive (growing) tumors, and the third of toremifene-treated responsive (regressing) tumors. DNA and RNA derived from normal tissues of the same experimental animals were also analyzed. In Southern blot analysis of genomic DNAs, 2 tumors out of 23 contained a new Xbal site in the Ha-ras gene, indicating a point mutation in the second nucleotide of codon 61. Both of these tumors belonged to the group that had not received toremifene. No amplifications of the Ha-ras or the neu genes were observed. Although greatly variable, the levels of Ha-ras mRNA were highest in untreated tumors, lower in toremifene-treated, unresponsive tumors and even lower in toremifene-treated, regressing tumors, corresponding approximately to the levels detected in normal liver and uterus of untreated animals. Expression of the neu mRNA was variable and considerably lower than that of Ha-ras mRNA. It was similar in all 3 groups and somewhat elevated than in several non-malignant control tissues. Localization of c-Ha-ras expression by in situ hybridization revealed a relatively even distribution of the mRNA throughout the mammary tissue. The results suggest that mechanisms other than activation of the c-Ha-ras or neu genes are important for progression and regression of DMBA-induced rat mammary carcinomas.     

BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors

ABSTRACT: BACKGROUND: Development of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis. METHODS: Mammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform. RESULTS: Tumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors. CONCLUSIONS: Some of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic changes identified in this work are at very small scales and thus may provide a more feasible basis for the identification of the target gene(s). Identification of the genes underlying these chromosome changes can provide new insights to the mechanisms of mammary carcinogenesis.     

Effects of the aromatase inhibitor 4-hydroxyandrostenedione and the antiandrogen flutamide on growth and steroid levels in DMBA-induced rat mammary tumors

Summary Using dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat as model, comparison was made of the effect of treatment for 20 days with the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) (7.5 mg, twice daily) or the antiandragen flutamide (5 mg, twice daily) on tumor growth as well as on plasma and tumor content of estrogens, androgens, and their precursors and metabolites. Tumor number and size were markedly decreased following treatment with either drug, the effect of treatment being more important on size than number, and on new tumors which developed during treatment than on tumors already present at start of treatment. Treatment with the aromatase inhibitor 4-OH-A caused a parallel decrease in plasma and tumor levels of pregnenolone (Preg), progesterone (P), and 17-OH P, while there was a marked increase in dehydroepiandrosterone (DHEA), androst-5-ene-3,17-diol (⁵-diol), androstenedione (⁴-dione), testosterone (T), androstane-3, 17-diol (3-diol), and androstane-3, 17-diol (3-diol), with no significant change in dihydrotestosterone (DHT) and 17-estradiol levels. The marked increase in tissue T content coupled to a decrease in P levels could well contribute to the inhibition of tumor growth induced by 4-OH-A. Flutamide, on the other hand, caused a marked fall in plasma and tissue levels of Preg, 17-OH Preg, P, and 17-OH P, with no significant change in the concentration of the other steroids, thus suggesting a possible role of the fall in tissue P levels in the inhibition of tumor growth. Since both drugs are potent inhibitors of DMBA-induced tumor growth in intact animals, better knowledge of their mechanism of action should add to our understanding of the multiple endocrine factors controlling the growth of these tumors.     

Dietary soy modulation of biochemical parameters in DMBA-induced mammary tumors

The aim of this study was to extend our previous observations on the soy modulation of biochemical parameters in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumors, by simultaneously investigating the expression of estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), progesterone receptor (PR), apoptosis, neu, and markers of cell proliferation, such as proliferating cell nuclear antigen (PCNA) by immunohistochemistry. The percentage of ERalpha positive tumors was 65.8% in masses from control animals, and significantly dropped to 36.8% in tumors from soy treated rats (P=0.010). The percentage of ERbeta positive tumors was 70.3% in masses from control animals vs. 50.0% in tumors from soy exposed animals (P=0.066). Moreover, the percentage of cases which were both ERalpha and ERbeta positive was significantly lower (17.6%) in soy treated than in control animals (51.3%) (P=0.006). The percentage of PR positive tumors was 34.2% in control animals vs. 2.6% in tumors from soy treated rats (P=0.0006). There were no statistically significant differences in the percentage of tumors positively stained for neu, apoptosis, or PCNA, in control vs. soy treated rats. However, when analyzing the reciprocal correlation among the different biochemical parameters we showed that, in treated animals, the majority of ERalpha positive tumors (91.7%) were also PCNA positive (P=0.036). The median percentage of PCNA positivity was significantly higher in ERalpha positive than in ERalpha negative tumors (25 vs. 5%) (P=0.0031). Moreover, an association was found between PCNA and neu status since all neu positive tumors were also PCNA positive (P=0.011).