In vivo differentiation and genomic evolution in adult male germ cell tumors.

Germ cell tumors (GCTs) are the most common solid malignancy in young adult men, but the genes and genomic regions involved in their etiology are not fully defined. We report here an investigation of DNA copy number changes in GCTs using 1 Mb BAC arrays. As expected, 12p gain was the defining genomic alteration, occurring in 72/74 GCTs. Parallel expression profiling of these tumors identified potential oncogenes from gained regions (LYN and RAB25) and potential tumor suppressor genes in regions of loss (SYNPO2, TTC12, IGSF4, and EPB41L3). Notably, we observed specific genomic alterations associated with histology, including gain of 17p11.2-q21.32 and loss of 2p25.3 in embryonal carcinoma, gain of 8p23.3-12 and loss of 5p15.33-35.3, 11q23.1-25, and 13q12.11-34 in seminoma, and gain of 1q31.3-42.3, 3p, 14q11.2-32.33, and 20q and loss of 8q11.1-23.1 in yolk sac tumors (YST). Many significant genes that mapped to these regions had previously been associated with specific histologies, such as EOMES (chr3) and BMP2 (chr20) in YST and SPRY2 (chr13) and SOX17 (chr8) in seminomas. Additionally, our results suggest a model in which histologic differentiation of GCTs may drive genomic evolution.

Resistance of Copenhagen rats to hepatocarcinogenesis does not involve T-cell immunity.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant, compared with susceptible F344 rats, to the growth of glutathione S-transferase 7-7 (GST 7-7) positive preneoplastic liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. Donryu rats, a strain with a level of susceptibility similar to F344, have a reduced T-cell response compared with the closely related, but highly resistant, DRH rat. Cop and DRH rats share several characteristics in their resistance to preneoplastic liver lesion growth and this study, therefore, was designed to examine whether T-cells play a role in Cop resistance. Cop rats were crossed with an athymic (nude) rat to produce F1s that were then interbred to produce F2 animals, some of which were nude with a partial Cop background. A comparison of the susceptibility of nude F2 animals and their euthymic (non-nude) littermates allowed us to determine what role, if any, T-cells play in Cop resistance. We treated 11 Cop, 11 F344, 19 nude F2s, and 18 non-nude F2s with diethylnitrosamine (DEN), followed 3 weeks later by a modified RH protocol. As expected, F344 rats were highly susceptible, having 41.9 +/- 3.3% (mean +/- SEM) of their liver section areas occupied by GST 7-7-positive lesions and Cop rats were highly resistant, having only 4.7 +/- 1.1% of their liver section areas occupied by lesions. Both nude and non-nude F2s were, like Cop rats, highly resistant (1.8 +/- 0.29 and 2.7 +/- 0.45%, respectively). These results show that T-cells are unnecessary for Cop rat resistance, or only play a minor role, and that the nude parental strain is also likely to be resistant to the growth of preneoplastic liver lesions.

Resistance to mammary tumorigenesis in Copenhagen rats is associated with the loss of preneoplastic lesions.

The resistance of Copenhagen (Cop) rats to mammary tumor development has recently been linked to three loci, but the genes have yet to be cloned and the mechanism of resistance is still largely unknown. In order to determine the cellular events associated with resistance, we prepared mammary whole mounts from Cop and susceptible Wistar Furth (WF) rats 0, 15, 30, 45 and 60 days after treatment with 50 mg/kg N-methyl-N-nitrosourea (MNU). At 15 days, treated rats of both strains had significantly more undifferentiated structures [terminal end buds (TEBs)] and significantly fewer differentiated structures [alveolar buds (ABs)] than untreated rats. Treated Cop rats, however, had significantly more TEBs and fewer ABs than age-matched, treated WF rats. Histological analysis of preneoplastic lesions tentatively identified from the whole mounts showed that like WF rats, Cop rats developed early preneoplastic lesions [intraductal proliferations (IDPs)] by 15 days post-MNU treatment. Unlike IDPs from WF rats, however, the IDPs in Cop rats then decreased in number until they were absent 60 days post-MNU treatment. Furthermore, they failed to progress into more advanced lesions such as ductal carcinomas in situ (DCIS). Finally, we found G-->A activating mutations in codon 12 of the Ha-ras gene in 60% of IDPs from Cop rats and 75% of IDPs from WF rats. Our results show that resistance in Cop rats is not due to a target cell population for the carcinogen that is smaller than in susceptible rats or to the failure of the carcinogen to inhibit mammary gland differentiation. Furthermore, we have shown that Cop rats develop preneoplastic IDPs that harbor Ha-ras mutations but, unlike IDPs in susceptible strains, they fail to progress and ultimately disappear.

Cyclin D1 expression during rat mammary tumor development and its potential role in the resistance of the Copenhagen rat.

BACKGROUND: Resistance to mammary tumorigenesis in Copenhagen rats is associated with loss of early preneoplastic lesions known as intraductal proliferations. The cause of this disappearance, however, is unknown. RESULTS: There were no differences in the numbers of lesions in mammary whole-mounts prepared from Copenhagen or Wistar-Furth rats at 20 or 30 days after N-methyl-N-nitrosourea treatment, but at 37 days there were significantly fewer lesions in Copenhagen glands. Furthermore, lesions in Copenhagen glands were exclusively intraductal proliferations, whereas in Wistar-Furth glands more advanced lesions were also present. Immunohistochemical staining showed frequent cyclin D1 overexpression in Wistar-Furth lesions at 37 days, but not in Copenhagen lesions. There were, however, no differences in p16INK4a protein expression, bromodeoxyuridine labeling and apoptotic indices, or mast cell infiltration between Copenhagen and Wistar-Furth lesions at any time. CONCLUSIONS: Overexpression of cyclin D1 in preneoplastic lesions may be important in the development of mammary tumors in susceptible rats, although this overexpression does not appear to cause significant changes in cell kinetics. Furthermore, the low levels of cyclin D1 expression in Copenhagen intraductal proliferations may play a role in the resistance of these rats to mammary tumorigenesis.

Resistance of Copenhagen rats to chemical induction of glutathione S-transferase 7-7-positive liver foci.

Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well-characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2-acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules.

Resistance to chemically-induced mammary tumors in Copenhagen X nude-derived F2 athymic rats: evidence that T-cell immunity is not involved in Copenhagen resistance.

Resistance to chemically-induced mammary tumors in the Copenhagen rat is well defined, but the mechanism of resistance has yet to be determined. We have tested whether or not Copenhagen rat resistance is dependent on T-cells, since several lines of evidence supported an involvement of the immune system. We crossed Copenhagen rats with an athymic nude rat to produce F1s, that were interbred to produce F2 animals, some of which were athymic with partial Copenhagen rat background. A comparison of the mammary tumor incidences between the nude athymic F2 animals and their non-nude littermates allowed us to determine what role, if any, T-cells played in resistance. Following treatment with N-methyl-N-nitrosourea, we observed no difference in the tumor incidences between the two groups. Furthermore, the mammary tumor incidences in the F2 nude and non-nude animals was almost zero. These results indicate that T-cells are not involved in Cop resistance, and that nude rats are resistant to N-methyl-N-nitrosourea-induced mammary tumorigenesis.