Genetic basis of sex-specific resistance to neuro-oncogenesis in (BDIX × BDIV) F2 rats

The identification of cancer susceptibility- and resistance-mediating genes is an essential prerequisite for prevention and early diagnosis of malignant tumors. Model organisms are helpful to identify variant alleles involved in pathways affecting individual cancer risk. BDIX and BDIV rats of both sexes are highly susceptible and resistant, respectively, to the development of N-ethyl-N-nitrosourea (ENU)-induced malignant peripheral nerve sheath tumors (MPNST), predominantly in the trigeminal nerves. Nevertheless, female (BDIV × BDIX) F₂ intercross rats have a lower MPNST incidence and a longer latency time than males. Six of seven autosomal gene loci (Mss1-Mss7) controlling genetic susceptibility and resistance in (BDIV × BDIX) F₂ hybrids exert allele- and sex-specific effects on tumor incidence and/or latency time of variable strength. Homozygous BDIV alleles at Mss4 or Mss7 located on rat chromosomes 6 and 10, respectively, are sufficient to cause almost complete resistance to ENU-induced MPNST development in female F₂ rats regardless of the genotype of the other locus. Both loci display only weak effects on male cancer risk. Survival curves of ENU-treated F₂ females depleted of animals with homozygous BDIV alleles at Mss4 and Mss7 are not significantly different from those of males, suggesting that these loci account mainly for the excess tumor resistance observed in female F₂ rats. By haplotype analysis Mss4 and Mss7 could be narrowed down to 20 and 12 Mb, respectively, providing a basis for the positional identification of candidate genes.     

High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters—the latency period, take rates, pathological features and metastatic rates—were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.     

Suppressive effect of an inducible nitric oxide inhibitor, ONO-1714, on AOM-induced rat colon carcinogenesis.

The expression of inducible nitric oxide synthase (iNOS) is markedly elevated in rat colon cancers induced by azoxymethane (AOM). In addition, iNOS can be detected in most adenomas and dysplastic aberrant crypt foci (ACF), suggesting that iNOS plays an important role in colon carcinogenesis. In the present study, the effect of an iNOS inhibitor, ONO-1714 ((1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0] heptane hydrochloride), on AOM-induced rat colon carcinogenesis was investigated. Male F344 rats were treated with 15 mg/kg body weight of AOM once a week, for 2 weeks. ONO-1714 was given to the rats at doses of 10, 20, 50, and 100 ppm in diet for 4 weeks from the day before the first carcinogen treatment. The number of AOM-induced ACF in the rats receiving 10, 20, 50 and 100 ppm ONO-1714 were 94, 73 (P < 0.05), 71 (P < 0.005), and 53% (P < 0.0005), respectively, of the control value. Moreover, the mean number of aberrant crypts per focus was significantly lowered in 100 ppm ONO-1714 group (P < 0.05). Then, the effects of long-term treatment (32 weeks) with 50 and 100 ppm ONO-1714 on AOM-induced colorectal tumor development were examined. Although incidences and multiplicities of colon tumors did not significantly differ among the groups, number of tumors developing in the middle part of colon were reduced with both 50 and 100 ppm doses (P < 0.05). Furthermore, colon tumor volume tended to be decreased by ONO-1714 treatment, and the number of colon tumors more than 3mm in diameter was significantly lowered in the 100 ppm ONO-1714 group (P < 0.01). These results suggest that iNOS plays roles in both early and late stages of colon carcinogenesis.     

The recombinant congenic strains—a novel genetic tool applied to the study of colon tumor development in the mouse

The development of tumors in mice is under multigenic control, but, in spite of considerable efforts, the identification of the genes involved has so far been unsuccessful, because of the insufficient resolution power of the available genetic tools. Therefore, a novel genetic tool, the RC (Recombinant Congenic) strains system, was designed. In this system, a series of RC strains is produced from two inbred strains, a background strain and a donor strain. Each RC strain contains a different small subset of genes from the donor strain and the majority of genes from the background strain. As a consequence, the individual genes of the donor strain which are involved in the genetic control of a multigenic trait, become separated into different RC strains, where they can be identified and studied individually. One of the RC strains series which we produced is made from the parental strains BALB/cHeA (background strain) and STS/A (donor strain). We describe the genetic composition of this BALB/cHeA-C-STS/A (CcS/Dem) series and show, using 45 genetic autosomal markers, that it does not deviate from the theoretical expectation. We studied the usefulness of the CcS/Dem RC strains for analysis of the genetics of colon tumor development. The two parental strains, BALB/cHeA and STS/A, are relatively resistant and highly susceptible, respectively, to the induction of colon tumors by 1,2-dimethylhydrazine (DMH). The individual RC strains differ widely in colon tumor development after DMH treatment; some are highly susceptible, while others are very resistant. This indicates that a limited number of genes with a major effect are responsible for the high susceptibility of the STS strain. Consequently, these genes can be mapped by further analysis of the susceptible RC strains. The differences between the RC strains were not limited to the number of tumors, but the RC strains differed also in size of the tumors and the relative susceptibility of the two sexes. Our data indicate that the number of tumors and the size of tumors are not controlled by the same genes. The genetics of these different aspects of colon tumorigenesis can also be studied by the RC strains. The DMH-treated mice of the parental strains and the RC strains also developed anal tumors and haemangiomas in varying numbers. The strain distribution pattern (SDP) of susceptibility for each of the three types of tumors induced by DMH is different, indicating that development of these tumors is under control of different, largely non-overlapping, sets of genes. Thus, with a single series of RC strains, genes involved in tumorigenesis in various organs and tissues can be studied separately. These results indicate that the novel genetic tool, the RC strain system, offers new possibilities for analysis of the multigenic control of tumor development.     

A comparison of succinyladenylate lyase activity and serum sialic acid as markers of malignancy

A comparison was made of succinyladenylate lyase (SAMP lyase), total serum sialic (TSA), and lipid soluble serum sialic acid (LSA) as early markers of malignancy in three experimentally induced rat tumor models. Elevation of SAMP lyase in 3'-methyl-dimethylaminoazobenzene-induced hepatic tumors at 2 weeks corresponded with microscopic detection of preneoplastic lesions with elevation of LSA occurring 2 weeks later. Elevation of breast SAMP lyase concurred with macroscopic presence of dimethylbenzanthracene involved breast tumors with elevation of LSA occurring 12 weeks later. Neither colon SAMP lyase nor LSA increased in rats bearing colon tumors induced by dimethylhydrazine. The determination of TSA was not a reliable indicator of tumor presence for the three types of tumors investigated. Both SAMP lyase and LSA are very good early indicators of hepatic tumor with SAMP lyase an earlier indicator of breast tumor than LSA.     

Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response—sequentially involving macrophages, T helper- and cytotoxic T lymphocytes—is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.     

Chronic exposure to a GSM-like signal (mobile phone) does not stimulate the development of DMBA-induced mammary tumors in rats: results of three consecutive studies

Certain epidemiological and experimental studies raised concerns about the safety of radiofrequency (RF) electromagnetic fields because of a possible increased risk of leukemia and lymphoma. In this study, an RF field used in mobile telecommunication was tested using 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats as a model for human breast cancer. Three experiments were carried out under strictly standardized conditions and were started on the same day of three consecutive years. The field consisted of a GSM-like signal (900 MHz pulsed at 217 Hz, pulse width 577 micros) of relatively low power flux density (100 microW/cm(2) +/- 3 dB) and was applied continuously throughout each experiment to freely moving animals. The specific absorption rates averaged over the whole body were 17.5-70 mW/kg. The highest values in young animals were at or around the exposure limit permissible for the general public (i.e. 80 mW/kg). The animals were palpated weekly for the presence of mammary tumors and were killed humanely when tumors reached a diameter of 1-2 cm to allow a reliable histopathological classification and a distinction between malignant and benign subtypes. The overall results of the three studies are that there was no statistically significant effect of RF-field exposure on tumor latency and that the cumulative tumor incidence at the end of the experiment was unaffected as well. The risk ratios were 1.08 (95% CI: 0.91-1.29) and 0.96 (95% CI: 0.85-1.07) for benign and malignant tumors, respectively. These observations are in agreement with other published findings. In the first experiment, however, the median latency for the development of the first malignant tumor in each animal was statistically significantly extended for RF-field-exposed animals compared to controls (278 days compared to 145 days, P = 0.009). No such differences were detected in the two subsequent experiments. These results show that low-level RF radiation does not appear to possess carcinogenic or cancer-promoting effects on DMBA-induced mammary tumors. To explain the mechanisms underlying the different results obtained in the three experiments, a hypothesis is presented which is based upon the neuroendocrine control mechanisms involved in the promotion of DMBA-induced mammary tumors. Despite the apparent absence of stimulatory effects of low-level RF-field exposure on the development and growth of solid tumors, it will be necessary to verify these results for leukemias and lymphomas, which may have completely different biological control mechanisms.     

Effect of intratumoral injection of carboplatin combined with pluronic P85 or L61 on experimental colorectal carcinoma in rats

Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on the intratumoral chemotherapy of an experimental adenocarcinoma in rats was examined. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histologic evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low-dose CPt (2.8 mg/kg) and P85 or L61 exhibited significant reductions in tumor volume after 28 days relative to Day 0 (112.7% +/- 34.4%, n = 15; 131.3% +/- 55.6%, n = 8) compared with tumors treated with free drug (339.4% +/- 75.0%, n = 16). Control tumors treated with either P85 or L61 alone or with saline showed volume increases of 1079.4% +/- 143.6% (n = 16), 729.4% +/- 202.2% (n = 7), and 1119.2% +/- 6.1% (n = 16), respectively. Treatment with high-dose CPt (20.7 mg/kg) led to a 79.3% +/- 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that 28.0 mg/kg of P85 significantly reduced levels of intracellular ATP to 44.7% +/- 1.5% of controls, whereas L61 at this concentration depleted ATP levels completely. Results confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma, leading to a significant reduction of tumor growth compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences.     

Senna and the formation of aberrant crypt foci and tumors in rats treated with azoxymethane.

Chronic use of anthraquinone laxatives has been blamed for the induction of habituation and the development of colonic cancer, but there are no definitive studies which have demonstrated this. To evaluate the carcinogenic potential of anthraquinones, the effect of long-term senna pod extract (SE) treatment on either healthy rats or rats treated with an initiating tumor agent (azoxymethane--AOM) has been studied. SE (30 and 60mg/kg), administered for 110 weeks, did not induce the development of aberrant crypt foci (ACF) and tumors in healthy rats. The development of ACF and tumors in rats treated with AOM were significantly reduced by SE (30 and 60 mg/kg). These results suggest that a chronic SE use does not predispose to colon cancer. By contrast, SE might exert an anti-tumoral activity on rat colon carcinogenesis.     

Abscopal effect of boron neutron capture therapy (BNCT): proof of principle in an experimental model of colon cancer

The aim of the present study was to evaluate, for the first time, the abscopal effect of boron neutron capture therapy (BNCT). Twenty-six BDIX rats were inoculated subcutaneously with 1 × 10⁶ DHD/K12/TRb syngeneic colon cancer cells in the right hind flank. Three weeks post-inoculation, the right leg of 12 rats bearing the tumor nodule was treated with BPA-BNCT (BPA-Boronophenylalanine) at the RA-3 nuclear reactor located in Buenos Aires, Argentina, at an absorbed dose of 7.5 Gy to skin as the dose-limiting tissue. The remaining group of 14 tumor-bearing rats were left untreated and used as control. Two weeks post-BNCT, 1 × 10⁶ DHD/K12/TRb cells were injected subcutaneously in the contralateral left hind flank of each of the 26 BDIX rats. Tumor volume in both legs was measured weekly for 7 weeks to determine response to BNCT in the right leg and to assess a potential influence of BNCT in the right leg on tumor development in the left leg. Within the BNCT group, a statistically significant reduction was observed in contralateral left tumor volume in animals whose right leg tumor responded to BNCT (post-treatment/pre-treatment tumor volume <1) versus animals who failed to respond (post/pre ≥1), i.e., 13 ± 15 vs 271 ± 128 mm³. In addition, a statistically significant reduction in contralateral left leg tumor volume was observed in BNCT-responsive animals (post/pre <1) vs untreated animals, i.e., 13 ± 15 vs 254 ± 251 mm³. The present study performed in a simple animal model provides proof of principle that the positive response of a tumor to BNCT is capable of inducing an abscopal effect.     

Effects of GSM-900 microwaves on DMBA-induced mammary gland tumors in female Sprague-Dawley rats

The aim of this investigation was to test the hypothesis that sub-chronic whole-body exposure to GSM-900 microwaves had an effect on tumor promotion and progression. Mammary tumors were induced by ingestion of a single 10-mg dose of 7,12-dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats (Ico:OFA-SD; IOPS Caw). In two independent experiments, DMBA-treated animals were divided into four groups: sham-exposed (16) and exposed (three groups of 16 animals). The specific absorption rates (SARs), averaged over the whole body, were 3.5, 2.2 and 1.4 W/kg in the first experiment (May-July) and 1.4, 0.7 and 0.1 W/kg in the second experiment (September-November). Exposure started 10 days after DMBA treatment and lasted 2 h/day, 5 days/week for 9 weeks. Animals were exposed to plane waves with the electric field parallel to the long axis of the animals. Body weight and the number, location and size of the tumors were recorded at regular intervals. Rats were killed humanely 3 weeks after the end of exposure. The results are negative in terms of latency, multiplicity and tumor volume. With regard to tumor incidence, in the first experiment there was an increase in the rate of incidence at 1.4 W/kg but less at 2.2 W/kg and none at 3.5 W/kg. Overall, these results, which are rather inconsistent, do not bring new evidence of a co-promoting effect of exposure to GSM-900 signals using the DMBA rat model.     

Most lung and colon cancer susceptibility genes are pair-wise linked in mice, humans and rats

Genetic predisposition controlled by susceptibility quantitative trait loci (QTLs) contributes to a large proportion of common cancers. Studies of genetics of cancer susceptibility, however, did not address systematically the relationship between susceptibility to cancers in different organs. We present five sets of data on genetic architecture of colon and lung cancer susceptibility in mice, humans and rats. They collectively show that the majority of genes for colon and lung cancer susceptibility are linked pair-wise and are likely identical or related. Four CcS/Dem recombinant congenic strains, each differing from strain BALB/cHeA by a different small random subset of ±12.5% of genes received from strain STS/A, suggestively show either extreme susceptibility or extreme resistance for both colon and lung tumors, which is unlikely if the two tumors were controlled by independent susceptibility genes. Indeed, susceptibility to lung cancer (Sluc) loci underlying the extreme susceptibility or resistance of such CcS/Dem strains, mapped in 226 (CcS-10 x CcS-19)F2 mice, co-localize with susceptibility to colon cancer (Scc) loci. Analysis of additional Sluc loci that were mapped in OcB/Dem strains and Scc loci in CcS/Dem strains, respectively, shows their widespread pair-wise co-localization (P = 0.0036). Finally, the majority of published human and rat colon cancer susceptibility genes map to chromosomal regions homologous to mouse Sluc loci. 12/12 mouse Scc loci, 9/11 human and 5/7 rat colon cancer susceptibility loci are close to a Sluc locus or its homologous site, forming 21 clusters of lung and colon cancer susceptibility genes from one, two or three species. Our data shows that cancer susceptibility QTLs can have much broader biological effects than presently appreciated. It also demonstrates the power of mouse genetics to predict human susceptibility genes. Comparison of molecular mechanisms of susceptibility genes that are organ-specific and those with trans-organ effects can provide a new dimension in understanding individual cancer susceptibility.     

A quantitative assay for experimental allergic encephalomyelitis in the rat based on permeability of spinal cords to 125I-human gamma-globulin.

We have developed a quantitative assay for experimental allergic encephalomyelitis (EAE) in the rat based on permeability of the spinal cord to 125I-human gamma-globulin (HGG). This assay is highly reproducible and eliminates many of the drawbacks of assaying for EAE on the basis of clinical and/or histologic criteria. Using the assay, we have shown a direct correlation between onset of histologic changes in the spinal cord and onset of permeability changes in the spinal cord. No rat without histologic lesions manifest permeability alterations, and all rats with histologic lesions did manifest increased permeability to 125I-HGG. Furthermore, strains of rats susceptible to EAE demonstrated permeability changes, whereas resistant rats did not. In addition, we demonstrated by permeability and histologic criteria that guinea pig myelin basic protein emulsified with incomplete Freund's adjuvant is encephalitogenic in the Lewis rat. We also demonstrated that recipients of passive transfer of sensitized cells develop permeability changes along with histologic lesions. We conclude that measuring permeability to 125I-HGG in the spinal cords of rats is a valid assay for EAE, and its improves upon current indices of EAE in that it is readily quantifiable.     

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.     

Immunologic suppression of carcinogenesis in spontaneously hypertensive rats (SHR) with T cell depression

A strain of spontaneously hypertensive rats (SHR) showed a selective depression of T cell functions brought about by aging. Conversely, this strain had a high NK cell activity as compared to other normal rat strains. This SHR strain was found to be much more sensitive to the carcinogenic activity of low doses of MCA than were WKA rats with normal T cell functions. Allogeneic thymus grafts almost completely restored the T cell functions of SHR, whereas injection of an immunopotentiator, NSP, enhanced NK cell activity and also caused a partial recovering of T cell functions. When immunologic restoration was achieved, generation of killer T cells to syngeneic SMT-5 tumor cells was induced and the cytotoxic activity of NK cells to K-562 cells was also enhanced. But the cytotoxic activity to the SMT-5 cells of NK cells and macrophages from the treated or untreated SHR was not detected. Allogeneic thymus grafts induced a significant transplantation resistance against a syngeneic SMT-5 tumor and injection of NSP enhanced only the survival days of the rats. Allogeneic thymus grafts also significantly suppressed the incidence of tumors induced by MCA, whereas the injection of NSP was not effective in the prevention of tumor development but was effective in prolongation of latency periods. These results support the hypothesis that immune surveillance mediated by T cells is an important mechanism for the control of tumor development.     

Establishment of two rat hepatoma cell strains produced by a carcinogen initiation,phenobarbital promotion protocol

Summary Two Fischer 344 rat hepatoma cell strains, JM1 and JM2, have been isolated from a primary hepatocellular carcinoma. Primary tumor formation was induced in a two-thirds partially hepatectomized rat by a single low dose (70 mg/kg of diethylnitrosamine followed by chronic phenobarbital administration (0.1 g/100 ml drinking water). The primary tumors were passed three times by subcutaneous implantation of tumor fragments into the inguinal region of syngeneic recipients. The fourth pass way by injection of tumor cells directly into the livers of recipient rats. Several weeks later, the tumor-containing rat livers were subjected to collagenase perfusion. Two cell lines emerged from tissue culture of the cells isolated by perfusion. Each cell line was cloned by serial dilution. Cells JM1 and JM2 were tumorigenic when injected into syngeneic rats. The tumors, which arose from injected cell strains, exhibited several characteristics of hepatocellular carcinoma. Morphology was examined by light and electron microscopy. Histochemical studies of JM1 and JM2 cells grown in vitro and in vivo were done. The levels of tyrosine aminotransferase and three microsomal enzymes of importance to drug and carcinogen metabolism were investigated. To our knowledge, this is the first report of cell strains derived from an initiation promotion protocol in rats. These studies were supported by Grants CA-30241 and CA-26904 from the National Institutes of Health, Bethesda, MD, and Collaborative Research Agreement 808549 from the Environmental Protection Agency.     

Insulin in aging and cancer: antidiabetic drug Diabenol as geroprotector and anticarcinogen

The effects of new antidiabetic drug Diabenol (9-beta-diethylaminoethyl-2,3-dihydroimidazo-(1,2-alpha)benzimidazol dihydrochloride) on life span and spontaneous tumor incidence in NMRI and transgenic HER-2/neu mice as well as on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats are studied. It is shown that treatment with the drug failed influence body weight gain dynamics, food and water consumption and the body temperature, slowed down age-related disturbances in estrous function and increased life span of all and 10% most long-living NMRI mice. The treatment with Diabenol inhibited spontaneous tumor incidence and increased the mammary tumor latency in these mice. Diabenol treatment slowed down age-related changes in estrous function in HER-2/neu mice, failed influence survival of these mice and slightly inhibited the incidence and decreased the size of mammary adenocarcinoma metastases into the lung. In rats exposed to 1,2-dimethylhydrazine, treatment with Diabenol significantly inhibited multiplicity of all colon tumors, decreased by 2.2 times the incidence of carcinomas in ascending colon and by 3.1 times their multiplicity. Treatment with Diabenol was followed by higher incidence of exophytic and well-differentiated colon tumors as compared with the control rats exposed to the carcinogen alone (76.3% and 50%, and 47.4% and 14.7%, respectively). Thus, the drug increases survival and inhibits spontaneous carcinogenesis in mice and inhibits colon carcinogenesis in rats.     

Mechanical analysis of intestinal contractility in a neonatal maternal deprivation irritable bowel syndrome rat model

The aims of the present study are to investigate biomechanical properties and provide mechanical analysis of contractility in ileum and colon in a neonatal maternal deprivation (NMD) irritable bowel syndrome (IBS) rat model. Mechanical testing was done on segments from ileum and colon in 25 IBS rats and 13 Control rats. Morphometric data were obtained from digitized images of the segments at no-load and zero-stress states. Pressure and diameter changes were measured during flow and ramp distensions under active and passive experimental conditions. Circumferential stresses (force per area) and strains (deformation) were computed with referenced to the zero-stress state. The contraction frequency was analyzed. Contraction thresholds and maximum contraction amplitude were calculated in terms of mechanical stress and strain. Compared with controls, the IBS rats had lower body weight (P < 0.01), smaller colonic opening angle (P < 0.05), higher colonic contraction frequency (P < 0.05 and P < 0.01) and lower contraction thresholds of pressure, stress and strain in both ileum and colon (P < 0.05 and P < 0.01). The maximum contraction pressure, stress and strain did not differ between IBS and Control groups (P > 0.05). In conclusion, the pressure, stress, and strain to evoke contractility in ileum and colon were lower whereas the frequency of induced colon contractions was higher in NMD IBS rats compared to normal rats. Furthermore, zero-stress state remodeling occur in colon in NMD IBS rats. Further studies on the association between intestinal biomechanical properties, hypersensitivity and afferent signaling in the IBS animal models are warranted.