Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.     

Spi-1 is a putative oncogene in virally induced murine erythroleukaemias

Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.     

Nonrandom loss of chromosome 15 in Syrian hamster tumours induced by v-Ha-ras plus v-myc oncogenes

Nonrandom chromosome rearrangements, observed in a variety of human and animal tumours, are associated in some cases with enhanced expression or deregulation of cellular oncogenes. Recently, it was shown that normal, diploid rodent cells are neoplastically transformed following transfection with two cooperating oncogenes, for example myc plus ras. However, the number of steps necessary to convert a normal cell into a malignant cell is unknown. If activation of two oncogenes is sufficient for tumorigenicity, tumours derived from diploid cells transformed by the transfected oncogenes may remain diploid or have only random chromosome alterations. We have performed cytogenetic analyses of tumours formed after transfection of Syrian hamster embryo cells with either v-Ha-ras plus v-myc DNAs or polyoma DNA alone. Whereas polyoma-induced, tumour-derived cells were diploid, tumours induced by v-Ha-ras plus v-myc oncogenes were monoclonal and had a nonrandom chromosome change, monosomy of chromosome 15. Thus, an additional change, loss of chromosome 15, is required for or is advantageous for tumorigenicity induced by v-Ha-ras plus v-myc oncogenes. These results suggest that the neoplastic progression of normal, diploid cells requires more than two steps under certain conditions.     

Viral transduction of c-myc gene in naturally occurring feline leukaemias

Feline leukaemia virus (FeLV) is epidemiologically associated with induction of the majority of lymphoid tumours of the domestic cat. However, about one-third of these tumours are devoid of exogenous virus or show evidence of virus integration only after tumour outgrowth. To help define the genetic mechanisms of feline lymphomagenesis we have explored here the possibility that cellular oncogenes (c-onc genes) are rearranged in tumour cell DNA. Of 16 FeLV-positive T-cell tumours among 31 naturally occurring lymphomas, 2 showed evidence of recombinant FeLV proviruses containing myc oncogene sequences. One of the two produced a transmissible myc-containing FeLV. In both cases c-myc and its surrounding DNA appeared unaltered. We believe that the association of myc with FeLV may result in its activation and play a part in the development of a significant fraction of cat T-cell lymphomas. Our findings contrast with studies of experimental induction of chicken lymphoma, in which myc activation occurs by retrovirus promoter insertion near c-myc (refs 3-5), rather than by incorporation into virus.     

Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats

Induction of mammary carcinomas in rats by a single exposure to a carcinogen during sexual development often involves malignant activation of the Ha-ras-1 locus. Each of the Ha-ras-1 oncogenes present in tumours induced by N-nitroso-N-methylurea, but not in those induced by 7,12-dimethylbenz(a)anthracene, became activated by the same G----A transition, the type of mutation induced by N-nitroso-N-methylurea. These results are consistent with the notion that Ha-ras-1 oncogenes are directly activated by the carcinogen during initiation of neoplasia.     

Identification of reciprocal translocation sites within the c-myc oncogene and immunoglobulin mu locus in a Burkitt lymphoma

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.     

Ha-ras oncogenes are activated by somatic alterations in human urinary tract tumours

DNA-mediated gene transfer (transfection) studies using NIH 3T3 cells as recipients have demonstrated the presence of transforming genes (oncogenes) in diverse human tumours. A large proportion of oncogenes so far detected by DNA transfection are related to the Ha-ras onc gene of Harvey (and BALB) murine sarcoma viruses (MSV), Ki-ras, the oncogene of Kirsten MSV, and a third member of the ras gene family, N-ras. Individual tumours of many different organs have been associated with the activation of members of the ras gene family. We now present the first systematic survey of human urinary tract tumours processed immediately after surgery, as well as normal tissues from the same patients, to detect the presence of such genes. We demonstrate activation of Ha-ras as an oncogene in around 10% of randomly selected urinary tract tumours as well as direct evidence that oncogene activation is the result of a somatic event which is selected for within the tumour cell population.     

A cellular oncogene (c-Ki-ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells

The cellular oncogene c-Ki-ras is amplified 30- to 60-fold in cells of the mouse adrenocortical tumour Y1. The amplified oncogene is located in double minute chromosomes and in a homogeneously staining chromosomal region, common karyotypical anomalies of tumour cells. The amounts of c-Ki-ras specific mRNA and of the protein (p21) encoded by the amplified gene are correspondingly elevated. Amplification and enhanced expression of cellular oncogenes may contribute to the genesis and/or maintenance of at least some naturally occurring tumours.     

Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer

Recently, we identified recurrent gene fusions involving the 5' untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers. Whereas TMPRSS2-ERG fusions are predominant, fewer TMPRSS2-ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3-13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5' fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3-14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5' fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.     

Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus

Proviruses cloned from rat cells infected with mouse mammary tumour virus, a B-type retrovirus regulated by glucocorticoid hormones, show the structural features of transposable elements: short inverted repeats conclude long direct repeats at the ends of viral DNA, and short sequences of cellular DNA are duplicated during integration and flank each provirus. The integrative mechanism joins a precise site in viral DNA to non-homologous sites in host DNA.     

Trisomy represses Apc(Min)-mediated tumours in mouse models of Down's syndrome

Epidemiological studies spanning more than 50 yr reach conflicting conclusions as to whether there is a lower incidence of solid tumours in people with trisomy 21 (Down's syndrome). We used mouse models of Down's syndrome and of cancer in a biological approach to investigate the relationship between trisomy and the incidence of intestinal tumours. Apc(Min)-mediated tumour number was determined in aneuploid mouse models Ts65Dn, Ts1Rhr and Ms1Rhr. Trisomy for orthologues of about half of the genes on chromosome 21 (Hsa21) in Ts65Dn mice or just 33 of these genes in Ts1Rhr mice resulted in a significant reduction in the number of intestinal tumours. In Ms1Rhr, segmental monosomy for the same 33 genes that are triplicated in Ts1Rhr resulted in an increased number of tumours. Further studies demonstrated that the Ets2 gene contributed most of the dosage-sensitive effect on intestinal tumour number. The action of Ets2 as a repressor when it is overexpressed differs from tumour suppression, which requires normal gene function to prevent cellular transformation. Upregulation of Ets2 and, potentially, other genes involved in this kind of protective effect may provide a prophylactic effect in all individuals, regardless of ploidy.