Oncogene database

26173

INTS1

integrator complex subunit 1;INTS1;integrator complex subunit 1

-

7p22.3

protein-coding

The proto-oncogene int-1 plays an important role in mammary tumorigenesis when activated by proviral insertions of the mouse mammary tumor virus. In normal mouse tissues the gene is expressed in the embryonic neural tube, suggesting a developmental function, while in Drosophila the homolog of int-1 is the segment polarity gene wingless. In order to study the protein products of int-1 we have derived fibroblast cell lines infected with multiple copies of a retroviral vector expressing int-1 cDNA. By Western blot analysis and immunoprecipitation we have identified a 44 kd form of int-1 protein which is secreted from these cells. The 44 kd species is distinct from the major intracellular forms of int-1 protein as judged by its slower mobility in SDS-polyacrylamide gels and by its longer half-life in pulse-chase experiments. Under normal growth conditions, little or none of the 44 kd protein is detectable in the cell culture medium but instead the majority is found associated with the extracellular matrix (ECM). The protein appears to bind heparin in vitro, suggesting that it might bind glycosaminoglycans in the ECM. These data support the view that int-1 protein may play a role in cell-cell communication over short distances.

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.

While there is convincing evidence implicating Drosophila int-1 in pattern regulation, the normal role of int-1 in vertebrate development is unclear. We have injected Xenopus eggs with mouse int-1 RNA and monitored subsequent development. Injected RNA is translated and the protein widely distributed. Embryos develop into apparently normal gastrulae, but almost ALL surviving neurulae have a bifurcated anterior and expanded posterior neural plate. Bifurcation of the neural plate was abolished by substitution of a single, conserved cysteine residue and was dependent on the presence of a signal peptide sequence in the int-1 protein. Histological examination indicates that underlying axial mesodermal structures were duplicated. This result suggests that ectopic int-1 expression leads to dual axis formation and points to a role for int-1 in patterning processes in vertebrate development.

DNA from nine different solitary subcutaneous lipomas, ALL with clonal abnormalities affecting the chromosome segment 12q13-15, was examined for rearrangement or amplification of a human INT1 gene sequence. INT1 is a putative oncogene that has been localized to the chromosome band 12q13. No rearrangement or amplification could be detected.

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of ALL but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.

The int-1 mammary oncogene is activated by proviruses of the Mouse Mammary tumor Virus in many different mammary tumors. We have inserted a genomic fragment containing the protein-encoding domain of the gene into the retroviral shuttle vector pZIPneoSV(X)1. After one round of virus replication we recovered recombinant proviral DNA containing a correctly spliced copy of int-1. In vitro transcription of this cDNA version of int-1 using SP6 polymerase and translation in a reticulocyte lysate yielded a protein of approximately 37,000 daltons. High expression of int-1 in NIH-3T3 cells infected with recombinant virus did not lead to morphological transformation.

The int-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.

We report the amplification of the putative oncogene INT1 in two of four retinoblastomas. In one case, the INT1 signal was amplified 10 to 15-fold, in the other 100-fold. In both cases there were signs of increased tumor aggressiveness with invasion of the choroid and development of metastases. The two cases without INT1 amplification had neither metastases nor locally invasive growth. Our findings indicate that INT1 amplification may be a feature of increased malignant potential in retinoblastomas.

The int-1 proto-oncogene encodes a primary product of 370 amino acids, is normally expressed in mid-gestational embryos and adult testis, and is activated by proviral insertions during mammary carcinogenesis. Polyclonal and monoclonal antibodies directed against int-1-specific synthetic peptides immunoprecipitate up to five forms of int-1 protein, ranging in size from 36,000 to 44,000 Mr, from cell lines that express cloned int-1 DNA introduced by transfection or infection with retroviral vectors. Pulse-chase labeling experiments and glycosidase digestions suggested that the smallest of the int-1 proteins is the primary translation product lacking its signal peptide and that it is modified to produce the larger species of sequential glycosylation. Subcellular fractionations demonstrated that ALL immunoprecipitable forms of int-1 are mainly associated with membranes. int-1 proteins in crude microsomal preparations are resistant to proteolysis and extractable at elevated pH, suggesting that they are sequestered within cytoplasmic vesicles in a manner consistent with the behavior of secretory products. However, we were unable to identify secreted int-1 products in extracellular fluids.

The int-1 proto-oncogene is the first cellular gene discovered and implicated in tumorigenesis solely on the basis of repeated insertional mutations that activate transcription of the gene. The gene is silent in most tissues but expressed in the embryonic central nervous system, in the late (post-meiotic) stages of spermatogenesis, and in a high proportion of mouse mammary tumor virus-induced carcinomas, when a provirus is inserted upstream or downstream of the coding domain. The functional significance of int-1 in the oncogenic process is supported by the demonstration that murine leukemia virus-based vectors carrying the gene can alter the morphology and growth properties of an established line of mammary epithelial cells. The predicted primary protein product of the int-1 gene is 370 amino acids in length and cysteine-rich; immunoprecipitation with anti-peptide antibodies reveals multiple species of int-1 protein, due to asparagine-linked glycosylations and probable cleavage of a signal peptide. However, the active product of the gene and its biochemical behavior during normal development and mammary tumorigenesis are not known.