Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product

The E7 transforming protein of human papilloma virus-16 binds to the retinoblastoma gene product (pRb) through a nine-amino-acid segment of E7 (21-29). This segment of E7 is homologous to the pRb-binding domains of the simian virus 40 large T and adenovirus E1A transforming proteins. Each of these viral transforming proteins bind to the same region of pRb. To isolate cellular proteins that interact with this viral protein-binding domain on pRb, we used recombinant pRb to screen a human complementary DNA expression library. Two cDNAs were isolated that encode retinoblastoma binding proteins (RBP-1 and RBP-2). We report here that these RBP genes exist in separate loci and produce discrete messenger RNAs. The predicted amino-acid sequence of these genes showed no homology to known proteins, but both RBPs contain the pRb binding motif conserved between E7, large T and E1A14. In vitro expression of the RBP cDNAs yielded proteins that specifically bound to pRb. Recombinant E7 protein, the E7 21-29 peptide and the homologous RBP-1 peptide inhibited RBP-pRb binding. Mutations introduced into the putative pRb-binding segment in RBP-1 impaired its binding activity. These studies indicate that the cellular RBP-1, RBP-2 and viral E7 proteins interact with pRb through similar domains.     

A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product

Tumour-suppressor genes, such as the human retinoblastoma susceptibility gene (Rb), are widely recognized as being vital in the control of cell growth and tumour formation. This role is indicated, in part, by the suppression of tumorigenicity of human tumour cells after retrovirus-mediated Rb replacement. How Rb acts to bring about this suppression is not clear but one clue is that the Rb protein forms complexes with the transforming oncoproteins of several DNA tumour viruses, and that two regions of Rb essential for such binding frequently contain mutations in tumour cells. These observations suggest that endogenous cellular proteins might exist that bind to the same regions of Rb and thereby mediate its function. We report here the identification of one such human cellular Rb-associated protein of relative molecular mass 46,000 (46K) (RbAP46). Two lines of evidence support the notion that RbAP46 and simian virus 40 T antigen have homologous Rb-binding properties: first, several mutated Rb proteins that failed to bind to T also did not associate with RbAP46; and second, both T antigen and T peptide (amino acids 101-118) were able to compete with RbAP46 for binding to Rb. The apparent targeting of the RbAP46-Rb interaction by oncoproteins of DNA tumour viruses strongly suggests that formation of this complex is functionally important.     

Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product

One of the cellular targets implicated in the process of transformation by the adenovirus E1A proteins is a 105K cellular protein. Previously, this protein had been shown to form stable protein/protein complexes with the E1A polypeptides but its identity was unknown. Here, we demonstrate that it is the product of the retinoblastoma gene. The interaction between E1A and the retinoblastoma gene product is the first demonstration of a physical link between an oncogene and an anti-oncogene.     

Human N-myc is closely related in organization and nucleotide sequence to c-myc

N-myc, a cellular gene related to the c-myc proto-oncogene, was originally identified on the basis of its very frequent amplification and overexpression in a restricted set of tumours, most notably human neuroblastomas. That N-myc may have a causal role in the genesis of these tumours is suggested by the observation that in the rat embryo fibroblast co-transformation assay it has a transforming potential similar to that of c-myc. The apparent structural and functional homology of N-myc and c-myc suggests that they may be members of the same protooncogene family. However, despite these apparent similarities, expression of the two genes appears to be dramatically different with respect to tumour specificity, as well as tissue and developmental stage specificity. To further elucidate the common and unique aspects of N-myc and c-myc gene structure and function in normal and transformed cells, we have determined the organization of human N-myc and the nucleotide sequence of its messenger product, and we report here that N-myc and c-myc have a similar intron/exon structure and that their protein products share regions of significant homology.     

Similarity of the product of the Drosophila neurogenic gene big brain to transmembrane channel proteins.

Cells in the neurogenic region of Drosophila embryos are initially bipotential; they can become either neuroblasts or epidermoblasts. Cell-cell interaction seems to play an important part in this developmental decision, which involves the function of a group of genes (the neurogenic genes). Loss-of-function mutations in any of the neurogenic genes result in nervous system hyperplasia and epidermal hypoplasia. Of the six known zygotic neurogenic genes, big brain (bib) is unique in several aspects. Most notably, all the other known neurogenic genes seem to fit into a cascade defined by genetic interactions, whereas bib does not show any detectable interaction with them. To understand how bib functions, we have now cloned the bib genomic and complementary DNAs. The predicted bib product shows significant sequence similarity to a family of transmembrane proteins, some of which form channels permeable to small molecules. Together with genetic studies, our results indicate that the bib product may mediate intercellular communication in a pathway separate from the one involving the products of the other neurogenic genes.     

Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture

Proto-oncogenes represent a group of eukaryotic genes whose activated forms are implicated in the development of cancer. We have recently identified a human gene, N-myc, that is distantly related to the proto-oncogene c-myc. N-myc is expressed at abnormally high levels consequent to amplification in numerous human neuroblastoma cell lines and metastatic neuroblastoma tumours. In addition, enhanced expression of N-myc, often a result of amplification, has been found in retinoblastoma cell lines and tumours (refs 5, 7 and M.S., unpublished data) and in cell lines derived from small-cell carcinomas of the lung. Here, we show that enhanced expression of N-myc subsequent to co-transfections of an N-myc expression vector and the mutant c-Ha-ras-1(EJ) (from the human bladder carcinoma cell line EJ) is a factor in tumorigenic conversion of secondary rat embryo cells. The transformed cells elicit tumours in athymic mice and isogeneic rats. The ability of N-myc to contribute to neoplastic transformation of cultured mammalian cells raises the possibility that enhanced expression consequent to amplification of N-myc may be a factor in the aetiology of human neuroblastoma.     

Parental origin of mutations of the retinoblastoma gene

Retinoblastoma and osteosarcoma arise from cells that have lost both functional copies of the retinoblastoma gene. Using the cloned retinoblastoma gene and other linked polymorphic loci, it is possible to reconstruct the sequential loss of the two homologous gene copies that precedes the development of these tumours. In non-hereditary tumours, the loss of each of the two homologues occurs somatically; in hereditary cases, the initial mutation is in the germline. Recently, Toguchida et al. reported that the paternally derived copy is preferentially the first one to become mutant during the genesis of non-hereditary osteosarcomas. We report here a similar analysis of patients with retinoblastoma in which we find no such predilection for initial somatic mutations. In contrast, when an initial mutation was a new germline mutation, it was derived from the father, a result which is consistent with new germline mutations arising primarily during spermatogenesis.     

Determination of spatial domains of zygotic gene expression in the Drosophila embryo by the affinity of binding sites for the bicoid morphogen

The maternal gene bicoid is a key component of the system that determines the pattern of the anterior half of Drosophila embryos. The bicoid protein forms a concentration gradient in early embryos, and is known to bind DNA. Specific binding sites are now shown to confer expression in a region of the embryo that depends on their affinity for bicoid protein: sites of high affinity allow expression further down the bicoid protein gradient than sites of low affinity.     

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.