MNU-induced Rat Research Articles

Biphasic effect of short-term pregnancy hormone treatment on N-methyl-N-nitrosourea-induced mammary carcinogenesis in young and old rats

This study examined the development of N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas in young and old female Lewis rats following short-term treatment with estrogen and progesterone to mimic pregnancy. Rats exposed at 4 weeks of age to MNU were treated at 6 weeks of age (early MNU/young E/P treatment) or at 24 weeks of age (early MNU/old E/P treatment) with a 21-day slow-release pellet containing 0.5 mg 17β-estradiol and 32.5 mg progesterone (E/P). Other rats were exposed to MNU at 22 and again at 23 weeks of age, and were treated with E/P at 24 weeks of age (late MNU/old E/P treatment). All experimental groups were compared with respective MNU-exposed age-matched E/P-untreated rats. Overt mammary carcinomas (≥1 cm in diameter) that were positive for hormone receptors were reduced in young E/P-treated rats, while hormone receptor-negative overt mammary carcinomas increased in old E/P-treated rats. The rate of development of small-sized mammary carcinoma (<1 cm in diameter) was similar in early MNU/young E/P-treated and late MNU/old E/P-treated groups, but higher in early MNU/old E/P-treated rats compared with respective E/P-untreated rats. At the termination of the experiment, normal mammary gland architecture had not been influenced by E/P treatment, although E/P treatment of older rats caused an increase in proliferating cell nuclear antigen (PCNA) labeling of the mammary tissue. Thus, the impact of short-term E/P treatment on MNU-induced rat mammary carcinogenesis is age-dependent and shows biphasic effects; the development of hormone-dependent overt mammary carcinomas was reduced in young rats but the development of hormone-independent overt mammary carcinomas increased in older rats. The enhanced outgrowth of hormone-independent overt mammary carcinomas by E/P treatment in old age is due to accelerated cell proliferation at the promotion/progression phase of mammary carcinogenesis. Age at short-term E/P treatment is crucial for breast cancer control.

Effects of novel retinoic acid metabolism blocking agent (VN/14-1) on N-methyl-N-nitrosourea (MNU)-induced mammary carcinoma and uterus in the rat model.

Abstract Abstract #2133 Background: All-trans-retinoic acid (ATRA) and other retinoids play key roles in prevention and therapy of many proliferative diseases including cancers. VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], which is a new generation novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of ATRA. The inhibitory effects of VN/14-1 on the growth of human breast cancer cells and human breast tumors in the nude mouse model have been previously demonstrated. The purpose of this study was to evaluate the effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, as well as on the uterus in immature ovariectomized (OVX) rats.&amp;#x2028; Methods: (1) VN/14-1 (5, 10, and 20 mg/kg/d) was given by oral gavage to grouped female Sprague Dawley (SD) rats bearing MNU-induced mammary carcinoma for 8 weeks, after which tumor weight and volume, as well as histology were measured. (2) VN/14-1 (10 and 20 mg/kg/d) and b-estradiol (10 mg/kg/d) were given alone or in combination, by oral gavage (VN/14-1) and subcutaneous injection (b-estradiol), to immature OVX SD rats for 3 days, after which uterine weight and histology were measured.&amp;#x2028; Results: (1) At the end of the treatment period, the administration of 5, 10 and 20 mg/kg/d VN/14-1 caused significant reductions of 19.1, 34.4 and 44.3%, respectively, in mean tumor weight compared with the control animals (all p &amp;lt; 0.05). The cumulative tumor growth was also significantly slower in groups receiving 5, 10 and 20 mg/kg/d compared to the control group in a dose-dependent manner. (2) Immature OVX rats given VN/14-1 at doses of 10 and 20 mg/kg, had reduction in uterine wet weight of up to 56% compared to OVX controls (P &amp;lt; 0.001). OVX rats given VN/14-1 of 10 and 20 mg/kg in combination with β-estradiol had reduction in uterine wet weight of up to 58% compared to the OVX rats given β-estradiol alone (P &amp;lt; 0.001). The adverse toxic effects such as fatigue and anorexia were occurred in the groups at high dose of 20 mg/kg.&amp;#x2028; Conclusions: RAMBA VN/14-1 was able to inhibit the growth of tumors in the MNU-induced ER positive rat mammary tumor model and antagonized the stimulatory effect of β-estradiol on the uterus. The studies suggest VN/14-1 might be an effective novel therapy for ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2133.

Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets

Rats treated with the alkylating agent methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. Roughly 50% of the tumors have Ha-ras mutation, whereas 50% do not. The MNU-induced rat mammary tumor model was employed to examine the therapeutic efficacy of the farnesyltransferase inhibitor (FTI), R115777, and to examine the use of genomics in identifying susceptible tumors as well as identifying genes whose expression are modulated by FTI treatment. In animals bearing palpable mammary tumors (< 7 mm diameter), we performed a surgical biopsy, and 3 days following the biopsy, rats were treated with R115777 (50 mg/kg body wt/day) by gavage. Tumors with Ha-ras mutations underwent profound regression, with nearly 90% showing complete regressions within 4 weeks. In contrast, the non-Ha-ras mutation-bearing tumors yielded a more variable response, although roughly half of the non-Ha-ras mutation tumors underwent significant regression. These results show that although all tumors appear to respond to the FTI inhibitor the tumors with Ha-ras mutations were exquisitely sensitive. We employed a microarray approach to define potential targets and the mechanism of action of R115777 in Ha-ras mutant or wildtype tumors following treatment with FTI. In addition, we determined whether gene expression prior to FTI treatment can be used to differentiate highly sensitive tumors (Ha-ras mutant) and tumors with variable sensitivity (Ha-ras wildtype). Untreated or FTI-treated (4 days at 50 mg/kg body wt) tumors (Ha-ras mutant or wildtype) were examined using oligonucleotide arrays. A significant number of genes were differentially expressed in control rat mammary tumors with or without an activated Ha-ras mutation, suggesting that a microarray analysis might differentiate highly sensitive and variably sensitive tumors. Most of the genes whose expressions were modulated by FTI in tumors were independent of Ha-ras status and were presumably modulated by effects on farnesylation of proteins other than Ha-ras. However, treatment of Ha-ras-mutated mammary tumors with R155777 results in preferential modulation of genes involved in ras-MAP kinase signal transduction pathway and in decreased expression of many genes involved with cell proliferation. In contrast, several classes of genes are altered in rat mammary tumors without a mutated Ha-ras, suggesting that non-ras targets are involved. Ras pathway related genes, p53, WT1 and PCNA, were preferentially modulated in Ha-ras-mutated tumors, whereas modulation of genes in the G-protein pathway, various cytochrome p450s and RB1 are involved in Ha-ras wildtype tumors. Elucidation of gene expression changes in FTI-treated or control rat mammary adenocarcinomas will help in identifying potential pharmacodynamic markers of FTI treatment as well as potential molecular targets of R115777 and other FTIs.

Nanoparticulate paclitaxel loaded into sterically stabilized mixed phospholipid micelles to improve chemotherapy of breast cancer

Active targeting of water-insoluble chemotherapeutic drugs, such as paclitaxel, to breast cancer is a highly desirable because of its associated increase in anticancer efficacy coupled with reduced systemic drug toxicity. However, rational design of these drug delivery platforms should take into account both pathobiological attributes of breast cancer, such as enhanced permeability and retention phenomenon and overexpression of vasoactive intestinal peptide (VIP) receptors, as well as biophysical properties of its ingredients, including ease of preparation, water insoluble drug loading capacity, steric hindrance, nanosize, and scale-up production and storage. To this end, we developed and tested a novel biocompatible and biodegradable nanoparticulate formulation of VIP-conjugated sterically stabilized phospholipid mixed micelles (SSMM-VIP; size ~ 14 nm) composed of disteraroyl phosphatidylethanolamnine-poly(ethylenglycol-2000) and egg yolk phosphatidylcholine. This construct solubilized 1 mg/ml paclitaxel (P-SSMM-VIP) and retained its biophysical properties upon lyophylization and reconstitution in saline. Moreover, it exhibited a twofold increase in cytotoxicity to MCF-7 breast cancer cells in comparison with P-SSMM and paclitaxel in DMSO (P < 0.05). In addition, the construct targeted VIP receptors overexpressed in methyl nitrosurea (MNU)-induced in situ rat breast cancer tissues. There was a twofold increase in accumulation of intravenously administered P-SSMM-VIP (1 mg/kg) in MNU-induced rat breast cancer, coupled with a significantly greater regression of breast cancer in comparison with P-SSMM and Taxol (P < 0.05). At the same time there was a significant reduction in P-SSMM-VIP accumulation in bone marrow, spleen and other organs in comparison with P-SSMM and Taxol (P < 0.05). There was no significant change in systemic arterial pressure during administration of P-SSMM-VIP. Collectively, these data indicate that actively targeting paclitaxel passively loaded into biocompatible, biodegradable, long-circulating SSMM to breast cancer through VIP receptors improves drug efficacy and reduces its uptake in injury-prone normal tissues. We suggest that P-SSMM-VIP is an efficacious and safe, actively targeted drug delivery platform to treat breast cancer.

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.

G1 Cell Cycle Regulatory Proteins in Chemically-Induced Rat Mammary Adenocarcinomas In Vivo and Tumor Promotion-Sensitive, -Resistant and Transformed Mouse Epidermal Cells In Vitro

Tumor promotion is characterized by selective proliferation of initiated cells resulting in their clonal expansion. Cyclin D1 is frequently upregulated in this process, but its expression does not necessarily correlate positively with cyclin A. In the present article, expression of G1 cell cycle regulatory proteins was systematically analyzed using two models of carcinogenesis: (a) N-methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas and normal rat mammary epithelial cells in vivo and (b) promotion- sensitive, -resistant, and transformed JB6 mouse epidermal cells in vitro. The results of this analysis revealed that p27Kip1 negatively correlated with cyclin D1. In addition, there were two types of correlations between p27Kip1 and cyclin A. First, p27Kip1 negatively correlated with cyclin A (type-I correlation). This scenario was observed in normal rat mammary epithelial cells in vivo and promotion-sensitive (P+) JB6 mouse epidermal cells, stimulated with phorbol ester (TPA) in vitro. Second, p27Kip1 positively correlated with cyclin A (type-II correlation). This correlation was observed in MNU-induced rat mammary adenocarcinomas in vivo and TPA-stimulated (P+) JB6 cells, treated with retinoic acid in vitro.

VIP receptors as molecular targets of breast cancer: implications for targeted imaging and drug delivery

Receptors for vasoactive intestinal peptide (VIP-R) are overexpressed in human breast cancer. This phenomenon may have important diagnostic and therapeutic implications because carrier systems loaded with imaging or therapeutic agents, and with surface ligands to VIP-R could potentially be actively targeted to breast cancer. Previously, we have prepared sterically stabilized liposomes (SSL) with VIP non-covalently associated on their surface. However, these liposomes were not able to actively target to breast cancer in rats in situ, most probably due to dissociation of non-covalently associated VIP from SSL. Hence, there is a need to conjugate VIP covalently to SSL. This study aims to begin to address this issue and to test the targeting ability of VIP–SSL to n-methyl nitrosourea (MNU)-induced rat breast cancer in vitro. First, VIP was conjugated to DSPE–PEG 3400–NHS [1,2-dioleoyl- sn-glycero-3-phosphoethanolamine- n-[poly(ethylene glycol)]- N-hydroxy succinamide, PEG M w 3400] under mild conditions to obtain a predominantly 1:1 conjugate of VIP and DSPE–PEG 3400 (DSPE–PEG 3400–VIP), as evidenced by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Next, DSPE–PEG 3400–VIP was inserted into preformed fluorescent cholesterol (BODIPY-Chol) labeled SSL by incubation at 37°C. To test breast cancer targeting ability in vitro, these VIP–SSL were subsequently incubated with MNU-induced rat breast cancer tissue sections. The results showed that when compared to fluorescent SSL without VIP or non-covalently attached VIP, significantly more VIP–SSL were attached to rat breast cancer tissues indicating that SSL with covalently attached VIP can be actively targeted to rat breast cancer tissues. This targeted carrier system is currently being explored for functional imaging and targeted chemotherapy of breast cancer.

EFFECTS OF EXERCISE ON ENDOTHELIAL FUNCTION IN AN MNU-INDUCED RAT MAMMARY TUMOR MODEL

The purpose of this study was to assess endothelial function in N-methyl-N-nitrosourea (MNU)-treated rats following eight weeks of moderate exercise training. A secondary purpose was to determine if exercise training altered vascular responsiveness to the chemotherapeutic agent 5-fluorouracil (5-FU). Virgin female Sprague Dawley rats were injected with MNU (50 mg/kg, i.p.) at 21 days of age, and at 28 days assigned to one of four groups: 1) non MNU + no exercise (NM+NE), 2) non MNU + exercise (NM+E), 3) MNU + no exercise (M+NE), 4) MNU + exercise (M+E). The exercise regimen consisted of treadmill running at 20–25 m/min, 15% grade, 30 min/d, 5 d/wk for 8 weeks. Vascular reactivity was evaluated in isolated aortic rings following 0, 4, and 8 weeks of exercise training. No significant differences in vascular responsiveness were noted between M+E and NM+E, or between M+NE and NM+NE animals at 0, 4, or 8 weeks. In addition, four weeks of exercise training resulted in no significant changes in vasoactivity for any group. However, 8 weeks of exercise training resulted in increased maximal endothelium-dependent vasorelaxation to acetylcholine (ACh, 1x10-5 M) (M+NE, 56 +/− 7%; M+E, 69 +/− 8%; p < 0.05) following norepinephrine-induced (1x10-7 M) vasoconstriction. Exposure of aortic rings from each group to 5-FU (7x10-3 M) resulted in vasoconstriction. Rings obtained from exercise trained animals demonstrated enhanced vasorelaxation to ACh (1x10-5 M) following 5-FU-induced vasoconstriction compared to rings obtained from untrained animals (p < 0.05). The results demonstrate that exercise training can enhance endothelium-dependent vasodilation in an MNU-induced rat mammary tumor model and that these changes may serve to counteract the vasoconstrictive properties of 5-FU. Supported by NIH KO7 CA79554

Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting.

Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with a wide range of biological activities. Receptors for VIP (VIP-R) are overexpressed in breast cancer, where they may have diagnostic and therapeutic implications. Although N-methyl nitrosourea (MNU)-induced breast cancer in rats is used extensively as a model to study mammary carcinogenesis, there is no information about the expression of VIP-R in this model. Therefore, the purpose of this study was to investigate the presence of VIP-R in MNU-induced breast cancer in rats so that this model can be used to perform studies involving VIP-R. Breast cancer was induced in 36-day-old virgin female Sprague-Dawley rats, by a single intravenous injection of MNU (50 mg/kg body weight). The breast tumors were detected 100-150 days after injection. The normal and cancerous rat breast tissue were excised and 20 micro sections were incubated with 40 nM fluorescein-labeled VIP (Fluo-VIP(TM)), in the presence and absence of 1000-fold excess unlabeled VIP, pituitary adenylate cyclase activating polypeptide (PACAP) or secretin. The sections were visualized under a fluorescence microscope and photographed. Fluo-VIP(TM) stained rat breast cancer tissue homogeneously and to a much greater extent than normal rat breast tissue (p < 0.05). This staining was specific as indicated by displacement of Fluo-VIP(TM) by excess unlabeled VIP and PACAP. Displacement of Fluo-VIP(TM) by secretin indicated the probable presence of VIP receptors of type VPAC1 (VIP receptor subtype 1) in the rat breast. These data suggest that, as in human breast cancer, VIP-R, predominantly of type VPAC1, are overexpressed in MNU-induced rat breast cancer tissue as compared to the normal rat breast tissue. Thus, MNU-induced rat breast cancer model can be used as a tool to study the functional role of VIP-R in human mammary carcinogenesis and VIP-R mediated active breast cancer targeting. This could have implications in the diagnosis, prognosis and therapy of human breast cancer.

Suppressive effects of chlorogenic acid on N-methyl-N-nitrosourea-induced glandular stomach carcinogenesis in male F344 rats.

The modifying effects of chlorogenic acid (CA) on N-methyl-N-nitrosourea (MNU)-induced glandular stomach carcinogenesis were investigated in five groups of male F344 rats. Rats in Groups 1 through 3 were given MNU in drinking water at a concentration of 400 ppm for 12 weeks. Animals of Group 1 were then kept on the basal diet alone, and those of Group 2 or 3 were fed a diet containing 500 or 250 ppm CA for a subsequent 22 weeks. Group 4 was exposed to CA alone through the experimental period (36 weeks), and Group 5 was given the basal diet continuously and treated as a control. At the end of the experiment, the incidence of glandular stomach carcinoma of Group 3 was significantly smaller than that of Group 1 (p < 0.03). The incidence of adenomatous hyperplasia of Group 2 was also significantly lower than that of Group 1 (p < 0.02). In addition, the proliferating cell nuclear antigen (PCNA) labeling index of the epithelial cells from the non-neoplastic mucosa in rats of Group 2 or 3 was significantly smaller than that of Group 1 (p < 0.0001). These results suggest that CA has a chemopreventive effect on MNU-induced rat glandular stomach carcinogenesis by exposure during the post-initiation phase, and CA may be a promising agent for prevention of human stomach cancer.

Defective Retrovirus Insertion Activates c-Ha-ras Proto-oncogene in an MNU-Induced Rat Mammary Carcinoma

Endogenous retrovirus sequences are present in the genome of a wide variety of animal species. The activation of the proto-oncogenes of the ras family, particularly c-Ha-ras, by either point mutation or overexpression, has been shown to be associated with a vast number of different cancers. Here we report that the insertion of a defective retrovirus in the −1 intron of rat c-Ha-ras is responsible for the activation of the gene by over 10-fold overexpression in an MNU-induced rat mammary cancer. A portion of the 3′ end of the retroviral sequence is expressed as a part of the c-Ha-ras transcript in the carcinoma tissue, indicating the direct involvement of this element in the transcription of the c-Ha-ras gene. The c-Ha-ras structural gene transcribed by the promoter of the defective retroviral element can neoplastically transform the NIH 3T3 cell line upon transfection.

Comparison of O6-methylguanine-DNA methyltransferase mRNA levels in Ha-ras mutated and non-mutated rat mammary tumors induced by N-methyl-N-nitrosourea.

Activation of the Ha-ras oncogene in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors has been well documented. Such Ha-ras activation is thought to be brought about by direct action of carcinogens resulting in a G-->A transition at the second nucleotide of codon 12. However, a DNA repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), can specifically remove methyl groups from O6-methylguanine, which is a major mutagenic and carcinogenic DNA lesion leading to the G-->A transition. In this study, we compared the amount of MGMT mRNA in MNU-induced rat mammary tumors with and without such Ha-ras activation. A single injection of MNU into 82 female Sprague-Dawley rats induced 80 mammary carcinomas. RNase protection analysis and subsequent sequencing revealed that 42 of 65 randomly selected tumors contained Ha-ras oncogenes activated by the G-->A transition. The amount of MGMT mRNA was then measured by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) amplification and Southern hybridization. No obvious difference in the level of MGMT mRNA was detected between the two tumor groups. In addition, in the course of our experiment, five of 42 tumors classified as containing activated Ha-ras oncogenes proved to contain low percentages of tumor cells with the Ha-ras activation. These results suggest that Ha-ras activation in MNU-induced rat mammary tumors may not necessarily be influenced by differences in MGMT activity. They also raise the possibility that activation of other oncogenes and/or inactivation of unidentified tumor suppressor gene(s) may be involved in development of a certain proportion of tumors with activated Ha-ras oncogenes, as is suspected in the case of tumors without Ha-ras activation.

Structure-activity relationships of four anti-cancer alkylphosphocholine derivatives in vitro and in vivo.

This study was carried out to investigate whether structure-activity relationships of alkylphosphocholines, a new group of anti-neoplastic agents, which had been detected in methylnitrosourea(MNU)-induced rat mammary carcinoma, can be transferred to in vitro systems. Therefore, the anti-neoplastic activity of 4 alkylphosphocholines (APCs) was compared in 6 tumor cell lines in vitro and in MNU-induced rat mammary carcinoma in vivo. The in vitro system consisted of 2 rat mammary-carcinoma-derived cell lines (1/C2 and 1/C32), as well as 2 human mammary-gland (MDA-MB-231 and MCF-7)- and gastrointestinal tract (HT-29 and KB)-derived tumor cell lines. As assessed by both cell counting and MTT-assay, the ranking of concentrations effecting 50% growth inhibition (IC50) was parallel in all cell lines for octadecylphosphocholine (18:0-PC), octadecenyl-(trans-9.10)-phosphocholine (t-18:1-PC) and octadecenyl-(cis-9.10)-phosphocholine (c-18:1-PC). Only hexadecylphosphocholine (16:0-PC) differed in its activity, being least active in 1/C2, 1/C32 and MDA-MB-231 cells, moderately active in KB and MCF-7 cells, and most active in HT-29 cells. The IC50 concentrations of APCs in the 2 rat mammary carcinoma cell lines significantly correlated with dosages effecting a 50% tumor growth delay in vivo. Remarkably, the 2 gastrointestinal cell lines were more sensitive to APC exposure than the mammary-carcinoma cell lines. In all cell lines except KB cells, growth-stimulation effects were seen in the concentration range preceding the anti-proliferative activity; in vivo, however, no accelerated cancer growth was observed. The in vitro system failed to describe the superior therapeutic ratio of c-18:1-PC, as assessed in vivo, because it does not take the relative sensitivity of tumor vs. normal cells into account. Complementary in vivo trials are therefore indispensable for a final evaluation. Comparison of the 2 in vitro assays shows good agreement of the interrelationship of IC50 values, those obtained by MTT assay being on average 25% higher than those obtained from cell counting.

Oncogene Alterations in Rat Colon Tumors Induced by N-Methyl-N-Nitrosourea

The authors assayed oncogene alterations in rat colon tumors induced by the direct-acting chemical carcinogen, N-methyl-N-nitrosourea (MNU). DNA isolated from 34 adenomas and eight carcinomas, as well as adjacent normal colon, of 11 rats was assayed by Southern blotting for restriction fragment length polymorphisms and gene amplifications and deletions in 13 oncogenes known to be involved in human or other animal tumors. In addition to finding apparent point mutations or other small alterations in the fos and abl genes in individual rat colon tumors, the authors observed what appear to be larger alterations (ie, rearrangements, or intragenic insertions or deletions) in the H-ras and myb loci in several tumors. In contrast, no changes in the K-ras, N-ras, myc, N-myc, neu, raf, fms, met, and hst genes were seen in any of these tumors. The frequency of myb gene alterations was higher in carcinomas than in adenomas, suggesting that these changes occurred relatively late during tumorigenesis and were not direct effects of the carcinogen. In addition, the finding of alterations in two or three oncogenes in several MNU-induced rat colon tumors suggests the possibility of more widespread genomic lesions in this model.

K-ras oncogene mutations in rat colon tumors induced by N-methyl-N-nitrosourea

We have been studying a rat model of colon cancer in which tumors are induced by direct application of N-methyl-N-nitrosourea (MNU) to discrete areas of the colonic mucosa for a limited period of time. Activation of the ras genes by point mutation has been observed in many experimental tumors, including tumors induced by MNU. To detect potential activating point mutations in the H-ras and K-ras oncogenes in MNU-induced rat colon tumors, DNA samples from 40 adenomas, nine carcinomas, and 14 histologically normal tissue samples from 14 rats--as well as from 16 foci induced on NIH3T3 cells by tumor DNAs--were amplified by the polymerase chain reaction and hybridized with allele-specific oligonucleotide probes. No H-ras point mutations were observed in any of these samples. We did detect K-ras point mutations, however, in four primary tumours--one adenoma (2.5%) and three carcinomas (33%); these mutations were all G----A transitions at the second nucleotide of codons 12 and 13. The absence of detectable ras mutations from the majority of tumors suggests that, in contrast to other animal models utilizing MNU, tumorigenesis in MNU-induced rat colon tumors may predominantly involve activation of genes other than ras.

Detection of transforming oncogenes in rat colon tumors induced by direct perfusion with N-methyl- N-nitrosourea

We assayed rat colon tumors induced by N-methyl-N-nitrosourea (MNU) for transforming oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA from 3 of 3 adenomas and 3 of 5 carcinomas induced transformed foci on NIH 3T3 cells. DNA from 2 of 3 primary foci also possessed focus-forming activity, and rat-specific sequences were observed in secondary focus DNAs. Furthermore, NIH 3T3 cells transfected with DNA from a carcinoma and from a primary focus derived from it, both positive in the focus-forming assay, induced tumors in nude mice. We found no evidence for rat H-ras, K-ras, or N-ras sequences in the DNA of any of 16 primary foci derived from 6 rat tumors; thus, in contrast to other animal tumor models induced by MNU, activation of the ras genes does not appear to predominantly occur in MNU-induced rat colon tumors. We also did not observe, in any of these foci, sequences corresponding to the rat neu, raf, fms, met, or hst genes, thus indicating that none of these is the transforming oncogene in our model. These results suggest that an as yet unidentified transforming oncogene may be activated in rat colon tumors induced by MNU.

Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma

Protein tyrosine kinase (PTK) activities in methyl nitrosourea (MNU)-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was almost equally distributed between the particulate and soluble (cytosolic) fractions at 110,000 X g. The activity of the particulate enzyme was stimulated by non-ionic detergent Triton X-100 by about 2-fold whereas the detergent had no effect on the cytosolic form. More than 60% of the particulate enzyme could be solubilized by 5% Triton X-100. Although, both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, poly (glu: tyr; 4:1) was the best substrate (apparent Km. 0.7 mg/ml). Both forms of enzymes utilized ATP as the phosphoryl group donor, with an apparent Km of 40 microM. Among various divalent cations tested, Co2+, Mn2+ and Mg2+ were able to fulfill the divalent cation requirement of both forms of the PTKs. All these cations exerted biphasic effects on the kinase activities, however, Mg2+ was the most potent cation. Agents such as epidermal growth factor, insulin and platelet derived growth factor which stimulate their respective receptor-PTK activities were without effect on PTK activities of mammary carcinoma. On the other hand, though heparin and quercetin inhibited both enzyme activities in a concentration dependent manner, the particulate form was more sensitive to inhibition than the cytosolic form. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms of PTKs. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play an important role in mammary carcinogenesis.

Absence of H-ras point mutation at codon 12 inN-methyl-N-nitrosourea-induced hepatocellular neoplasms in the rat

In order to assess the possibility that activated ras-associated hepatic carcinomas might be much rarer in rats than mice because of the more frequent or rapid occurrence of powerful carcinogenic event(s) other than ras point mutations in the former animals, precancerous lesions and hepatocellular carcinomas induced by a weak hepatocarcinogen N-methyl-N-nitrosourea (MNU) in the rat liver were analyzed for the presence or absence of ras point mutations. MNU was chosen because it is well known that MNU-induced rat mammary carcinomas contain activated H-ras at very high frequency. Male Fisher rats were treated with a single dose of MNU after partial hepatectomy, and then administered dietary phenobarbital or repeated s.c. injections of carbon tetrachloride as promoting procedures. Analyses by oligonucleotide hybridization, MnlI-restriction-fragment-length polymorphism and NIH3T3 cell transfection assays revealed neither H-ras point mutations nor transforming ability of the DNA from 36 MNU-induced rat hepatic neoplasms. The results were in agreement with previous results for rat hepatocellular carcinomas induced by other potent liver carcinogens and did not support our hypothesis that the frequency of finding ras activation might be dependent on the strength of the carcinogen.

Therapeutic effect of N-(4-hydroxyphenyl)retinamide on N-methyl- N-nitrosourea-induced rat mammary cancer

Virgin Sprague-Dawley rats received a single i.v. injection of 40 mg N-methyl-N-nitrosourea (MNU)/kg body wt. at 50 days of age. After the first palpable mammary tumor reached 10 mm in size, the animals were sequentially allocated to one of 4 groups: (I) placebo diet, (II) 10 μg tamoxifen s.c. 3 times per week, (III) 3 mmol N-(4-hydroxyphenyl)retinamide (4-HPR)/kg diet, or (IV) both (II) and (III). Weekly measurements of initial tumors and subsequent tumors were made throughout the study. 4-HPR administration resulted in a complete regression (non-palpable state) of the first mammary tumor in 6 animals (22%) and partial regression or non-progression in 5 others (19%). Tamoxifen alone induced only partial response in 9 animals (33%). 4-HPR and tamoxifen resulted in 19% total and 26% partial response. The data suggests therapeutic value of 4-HPR in MNU-induced rat mammary carcinoma.

New cytostatics with experimentally different toxic profiles

The narrow therapeutic ratio ofcytotoxic agents is a major drawback in the treatment of cancer because it limits the use ofanticancer chemotherapy. The underlying reason is the low affinity and specificity of the majority of anticancer agents to cancer cells which consequently damage neoplastic and normal cells to a similar extent. An interesting approach to overcoming this problem is based on qualitative or quantitative differences in enzyme levels in normal and neoplastic cells. A difference in the level of an O-alkyl cleavage enzyme which is barely detectable in a variety of tumor cells, but displays considerable activity in liver cells (16, 19), has led to the development of alkyllysophospholipids (ALP) as anticancer agents (12). They represent a class of compounds which obviously combine cytotoxic mechanisms of action by accumulating in phospholipid bilayers which in turn leads to the destruction of cancer cell membranes because these cells are unable to metabolize ALPs. They are able, moreover, to challenge the immune system (2, 12). For preclinical evaluation, the methylnitrosourea-induced autochthonous mammary carcinoma has been found to be a reliable and valid model in the selection of active compounds of this class of agents (5). Two interesting ALPs ( 1-octadecyl-2-methoxyrac-glycero-3-phosphocholine and 1-hexadecylmercapto-rac-methoxymethyl-3-propylphosphocholine) have been developed to Phase II clinical trials (3, 10). In a study on the structure-activity relationship of some newly synthesized compounds of this class that had been selected for their property to be cleaved by the O-alkyl-cleavage enzyme, an alkylphosphocholine (hexadecylphosphocholine, HPC) was used as 'negative control'. Remarkably, this compound had the highest antineoplastic activity of the whole series against MNU-induced rat mammary carcinoma. This result prompted a broader study of the antineoplastic activity o[ alkylphosphocholines which comprised experiments on the dose-response and structureactivity relationships of HPC and its congeners tetradecyclphosphocholine (TPC) and octadecylphosphocholine (OPC) (see Figure 1) in MNU-induced mammary carcinoma. It also included experiments on the anticancer efficacy of HPC against chemically-induced, autochthonous fibrosarcoma and colorectal carcinoma in rats. In addition, the toxicity of HPC in rats was examined with special emphasis given to the bone marrow toxicity using clonogenic stem cell assays.