Insertional mutagenesis by replication-deficient retroviral vectors encoding the large T oncogene.

Insertion sites of replication-deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor-suppressor proteins p53 and Rb by virtue of a chaperone-like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg-induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics ( approximately 8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D-derived monocytic leukemias showed hits in Pim2 and Max proto-oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector-mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.

Cryopreservation and orthotopic transplantation of rat ovaries as a means of gamete banking.

Besides the exponentially increasing number of mouse strains, the rising number of rat strains, due to the establishment of transgenic and coisogenic strains in this species, surpasses the capacity of most animal houses. Cryopreservation of gametes may be a means of solving these problems. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. Ovaries of the rat inbred strain WKY/Ztm were frozen with dimethylsulphoxide as cryoprotectant and stored at -196 degrees C. Orthotopical transplantation was performed into ovariectomized syngenic recipients. Re-establishment of the reproductive cycle in the recipients was confirmed by vaginal cytology. The morphological integrity of frozen and unfrozen ovaries was compared by histological means after staining with haematoxylin and eosin. The number of litters and offspring was recorded. Reproductive cycle was re-established in all recipients of unfrozen ovaries and in more than 50% of recipients that received frozen-thawed ovaries. One-third of the former and more than 25% of the latter became pregnant and delivered at least one litter. Cyropreservation of ovaries can thus be considered as a reliable method of preserving scientifically and economically important mutant stock, as well as congenic rat strains that are currently not required.

Contrasting effects of telomere shortening on organ homeostasis, tumor suppression, and survival during chronic liver damage.

Telomere shortening limits the regenerative capacity of cells during aging and chronic disease but at the same time inhibits tumor progression, and it has yet to be determined which of these mechanisms is dominantly affecting organismal survival. Here we show that telomere shortening in telomerase knockout (mTERC-/-) mice in combination with chronic liver damage significantly reduced organismal survival even though telomere shortening strongly inhibited liver tumor formation. Decreased survival induced by telomere shortening correlated with an imbalance between liver cell proliferation and liver cell apoptosis. Specific changes in gene expression were associated with telomere shortening and chronic liver damage and these gene expression changes were partially reversed by adenovirus mediated telomerase gene delivery. This study gives experimental evidence that the negative impact of telomere shortening on organ homeostasis and organismal survival can surpass the beneficial effects of telomere shortening on suppression of tumor growth in the setting of chronic organ damage.

Nephroblastomatosis and loss of WT1 expression associated with trisomy 13.

Trisomy 13 (Patau's syndrome) is a rare finding in newborns. The life span of babies affected by this chromosome abnormality is severely shortened, and multiple, partly severe malformations occur. In this study, we report on an unborn with trisomy 13 (artificially aborted on the 24th week) which showed, among other typical deformities, bilateral nephrogenic rests (nephroblastomatosis). Using molecular analysis, a loss of Wilms' tumor gene 1 (WT1) transcript and a biallelic expression of insulin growth factor 2 (IGF2) could be revealed. To our knowledge, this is the first reported case of trisomy 13 which showed this type of anomaly and gene expression findings.

Significant differences in the effects of magnetic field exposure on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in two substrains of Sprague-Dawley rats.

We have shown previously (S. Thun-Battersby et al., Cancer Res., 59: 3627-3633, 1999) that power-line frequency (50-Hz) magnetic fields (MFs) at micro T-flux densities enhance mammary gland tumor development and growth in the 7,12-dimethylbenz(a)anthracene (DMBA) model of breast cancer in female Sprague-Dawley (SD) rats. We also demonstrated that MF exposure results in an enhanced proliferative activity of the mammary epithelium of SD rats (M. Fedrowitz et al., Cancer Res., 62: 1356-1363, 2002), which is a likely explanation for the cocarcinogenic or tumor-promoting effects of MF exposure in the DMBA model. However, in contrast with our data, in a similar study conducted by Battelle in the United States, no evidence for a cocarcinogenic or tumor-promoting effect of MF exposure was found in the DMBA model in SD rats (L. E. Anderson et al., Carcinogenesis, 20: 1615-1620, 1999). Probably the most important difference between our and the Battelle studies was the use of different substrains of SD rats; the United States rats were much more susceptible to DMBA than the rats used in our studies. This prompted us to compare different substrains of SD outbred rats in our laboratory in respect to MF effects on cell proliferation in the mammary gland, susceptibility to DMBA-induced mammary cancer, and MF effects on mammary tumor development and growth in the DMBA model. The SD substrain (termed "SD1") used in all of our previous studies was considered MF-sensitive and used for comparison with another substrain ("SD2") obtained from the same breeder. In contrast with SD1 rats, no enhanced cell proliferation was determined after MF exposure in SD2 rats. MF exposure significantly increased mammary tumor development and growth in SD1 but not SD2 rats. These data indicate that the genetic background plays a pivotal role in effects of MF exposure. Different strains or substrains of rats may serve to evaluate the genetic factors underlying sensitivity to cocarcinogenic or tumor-promoting effects of MF exposure.

Lack of effects of sodium 2-mercaptoethane sulfonate (mesna) on Ochratoxin A induced renal tumorigenicity following life-time oral administration of Ochratoxin A in DA and Lewis rats.

Sodium 2-mercaptoethane sulfonate (Mesna) reacts with urotoxic metabolites of oxazaphosphorine drugs (e.g. cyclophosphamide or ifosfamide) and has been used clinically to protect against damage induced by these aggressive anti-neoplastic drugs in the kidney and lower urinary and genital tracts. Ochratoxin A (OTA) is a potent nephrotoxin in several species. In order to elucidate whether mesna has curative or preventive effects on OTA-induced renal damage or renal tumor development, we administered OTA and/or mesna to both DA and Lewis rats for their life-time and examined kidney, urethra and urinary bladder histologically. OTA induced sex- and strain-specific renal tumors. However, there was no evidence of any effect of mesna on the incidence and distribution of any type of tumor or non-neoplastic finding in the kidney in either strain or treated group. In this study, we have confirmed that mesna treatment did not show any curative or preventive effects on either OTA-induced kidney damage or renal tumor development in two different strains that have distinct metabolic characteristics.

TSPY expression is variably altered in transgenic mice with testicular feminization.

TSPY (testis-specific protein, Y-encoded) genes are expressed in premeiotic germ cells and round spermatids. The topology and timing of TSPY expression, and also its homology to members of the TTSN-family, suggest that TSPY is a proliferation factor for germ cells. There is also evidence for a role of TSPY in the aetiology of testis cancer. TSPY is a candidate for GBY, the elusive gonadoblastoma locus on the human Y chromosome, which is thought to predispose dysgenetic gonads of 46, XY sex-reversed females to develop gonadoblastoma. We have previously generated a TSPY transgenic mouse line (Tg(TSPY)9Jshm) that carries approximately 50 copies of the human TSPY gene on the mouse Y chromosome. In order to elucidate TSPY expression under complete androgen insensitivity and to investigate a possible role of TSPY in gonadal tumorigenesis, we have now generated sex-reversed TSPY transgenic Ar(Tfm) mice hemizygous for the X-linked testicular feminization mutation (Ar(Tfm)). We can show that the TSPY transcript is aberrantly spliced in the testes of TSPY-Ar(Tfm) mice, and that TSPY expression is upregulated by androgen insensitivity in some but not all animals. TSPY transgenic mice showed significantly increased testes weights. In one TSPY transgenic Ar(Tfm) animal, spermatogenesis proceeded beyond meiotic prophase. No tumors of germ cell origin were found in the testes of TSPY-Ar(Tfm) mice. Five out of 46 TSPY transgenic Ar(Tfm) mice, and 3 out of 31 age-related NMRI-Ar(Tfm) controls developed Leydig cell tumors, whereas none of the age-matched Ar(Tfm) mice (n=44) on a wild type background were affected by Leydig cell tumorigenesis.

Telomerase deletion limits progression of p53-mutant hepatocellular carcinoma with short telomeres in chronic liver disease.

BACKGROUND & AIMS: During early stages of carcinogenesis most human epithelial cancers including hepatocellular carcinoma (HCC) have been observed to transit through a "crisis" stage characterized by telomere shortening, loss of p53 checkpoint function, and a sharp increase in aneuploidy. The function of telomerase during in vivo hepatocarcinogenesis has not been studied in this genetic context. METHODS: Here we generated a mouse model in which HCC was induced by chronic organ damage (HBs-AG transgene) in the presence of telomere shortening and p53 deletion. Tumor development was analyzed in late-generation telomerase knockout mice (mTERC(-/-)) and littermates, genetically rescued for telomerase gene expression (mTERC(+/-)). RESULTS: The formation of HCCs was strongly suppressed in mTERC(-/-) mice compared to mTERC(+/-) siblings correlating with reduced rates of tumor cell proliferation and elevated rates of tumor cell apoptosis. Although the prevalence of short telomeres was similar in chronically damaged liver of both cohorts, mTERC(-/-) HCC developed increased levels of DNA damage and aneuploidy compared to mTERC(+/-) HCC. CONCLUSIONS: This study provides direct evidence that telomerase is a critical component for in vivo progression of p53 mutant HCC with short telomeres in the chronically damaged liver. In this molecular context, telomerase limits the accumulation of telomere dysfunction, the evolution of excessive aneuploidy, and the activation of p53-independent checkpoints suppressing hepatocarcinogenesis.

Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

Inhibition of lupus disease by anti-double-stranded DNA antibodies of the IgM isotype in the (NZB x NZW)F1 mouse.

OBJECTIVE: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing pathogenic IgG anti-double-stranded DNA (anti-dsDNA) autoantibodies are deposited in renal capillaries and initiate glomerulonephritis (GN) by the activation of complement and effector cells. In contrast, it has been demonstrated that the presence of IgM anti-dsDNA antibodies correlates negatively with the development of GN in SLE. The aim of this study was to determine whether anti-dsDNA antibodies of the IgM isotype protect against IC-mediated organ damage in SLE. METHODS: Lupus-prone (NZB x NZW)F(1) mice (females) were treated with murine monoclonal IgM anti-dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 mug/week starting at 16 weeks of age (prophylactic) or at 24 weeks of age (therapeutic). RESULTS: Mice treated with IgM anti-dsDNA exhibited a delayed onset of proteinuria and a reduced degree of renal pathology, which resulted in significantly improved survival as compared with control mice. Serum concentrations of IgG anti-dsDNA antibodies were not significantly modified. However, glomerular deposition of ICs was markedly reduced in both treatment protocol groups. In contrast, higher amounts of IgG and IgM and increased expression of Fcgamma receptor were demonstrated in liver sections from the treated mice compared with the untreated mice, suggesting an enhanced clearance of soluble ICs from phagocytic cells of the reticuloendothelial system. CONCLUSION: These data demonstrate the efficacy of IgM anti-dsDNA treatment in inhibiting the pathologic changes of lupus in (NZB x NZW)F(1) mice. Lower glomerular IC deposition is associated with a reduced inflammatory response and impaired organ damage. The reduced frequency of GN in SLE patients who have IgM anti-dsDNA antibodies may therefore reflect a disease-modifying effect of this class of autoantibodies that has potential therapeutic implications. Our findings should encourage the development of new therapeutic modalities using IgM anti-dsDNA antibodies in humans with SLE.

HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.

Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.

Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Strain-specific mammary proliferative lesion development following lifetime oral administration of ochratoxin A in DA and Lewis rats.

OTA, a potent nephrotoxin in several species, is a renal carcinogen in animals and is implicated in the etiology of BEN. The NTP classified OTA as having clear evidence of carcinogenic activity, based on uncommon tubular adenomas and tubular cell carcinomas of the kidney and multiple fibroadenomas of the mammary gland, seen in the rat. As shown previously (Castegnaro et al., Int J Cancer 1998;77:70-5), induction of renal tumors by OTA is sex- and strain-specific in DA and Lewis rats, with DA males being most responsive and DA females being resistant; however, that report was confined to the kidney and urinary tract. To obtain OTA-induced tumorigenic information in rats, we administered OTA (0.4 mg/kg) by oral gavage to both DA and Lewis rats for their lifetimes and extended the investigation to complete histopathology of all tissues and organs. We also observed the characteristic renal tumor that is highly strain- and sex-specific, and there were increased incidences of proliferative mammary lesions in Lewis rats but not in DA rats, indicating that these were also strain-specific. In view of the NTP report of OTA treatment-related mammary fibroadenoma in F344 rats, we observed increased mammary proliferative lesions in Lewis rats but not in DA rats. Our results suggest that OTA may play some role in mammary tumor development in some rat strains.