The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques. Previous results indicate that complete inactivation of the RB gene is required for tumour formation. As a 'cancer suppressor' gene, RB thus functions in a manner opposite to that of most other oncogenes. Sequence analysis of RB complementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function. To further substantiate and identify the RB protein, we have prepared rabbit antisera against a trypE-RB fusion protein. The purified anti-RB IgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000-114,000. The specific protein(s) are present in all cell lines expressing normal RB mRNA, but are not detected in five retinoblastoma cell lines examined. The RB protein can be metabolically labelled with 32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that the RB gene product may function in regulating other genes within the cell.

Mutational inactivation of the retinoblastoma gene (RB) is found in all retinoblastomas and in a subset of other human neoplasms, including sarcomas of bone or soft tissue and carcinomas of lung or breast. Exogenous copies of wild-type RB have been shown to suppress the tumorigenicity of several types of tumor cells with endogenous RB mutations, including a previously described human prostatic carcinoma cell line. To further support a role for RB inactivation in the genesis ofprostate cancer, seven primary or metastatic prostate carcinoma specimens were examined for evidence of RB mutation. By the use of immunoblot analysis and immunostaining of histologic sections, RB-encoded protein was readily detected in tumor cells offive specimens, was equivocally detected in one specimen, and was apparently absent from tumor cells of one specimen. RB mutations in the latter case were precisely characterized as (I) a deletion of 103 nucleotides containing transcriptional start sites and (it) loss of the second RB allele.The 103-base-pair deletion was sufficient to abolish the promoter activity of upstream DNA sequences in a heterologous expression system. These results (i) demonstrate thatRB can be inactivated in vivo by mutation of its promoter, (ii) confirm the existence ofRB mutations in some human prostate carcinomas, and (iii) suggest the use of immunohistochemical methods to screen for RB mutations in clinical samples of common adult neoplasms. mutations, as predicted by Knudson (9). RB mutations are also found in a subset of osteosarcomas, soft-tissue sarcomas, and carcinomas of breast and lung, suggesting a broad role for RB inactivation in the genesis of human tumors (8).Finally, restoration of the normal RB gene product, ppllORB, into pp11ORB-deficient (RB-) tumor cells alters several aspects of their neoplastic phenotype, including suppression of tumorigenicity in nude mice (10, 11). Because of these properties, RB is a model for studying other candidate tumor suppressor genes.In a previous study of three human prostate carcinoma cell lines, one was found to express a mutated RB mRNA and an abnormally small RB protein that was functionally inactive in tumor suppression (11). To establish the existence of RB mutations in native prostate carcinomas, primary or metastatic tumor specimens were screened for loss of RB protein expression. Two tumors were found to have severely reduced or undetectable amounts of RB protein by immunoblot analysis and by immunostaining of histologic sections. Direct detection of mutated RB alleles is hindered by the large size (';200 kilobases) and structural complexity (27 exons) of this gene (12). Nevertheless, a detailed genetic analysis of one tumor revealed both mutations leading to RB inactivation: (i) a 103-base-pair (bp) deletion that abolished activity of the RB promoter and (ii) loss of the second normal RB allele. Prostate carcinoma is the most common cancer in men (1).

Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans >60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G+C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G+C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern.

The amino acid sequence of the gene for the peplomer protein of the vaccine strain M41 and the Beaudette laboratory strain M42-Salk of avian infectious bronchitis virus (IBV) have been derived from cDNA sequences. As found with other coronaviruses, the peplomer protein carries the epitopes eliciting neutralizing antibodies. The gene encodes a primary translation product of 1162 amino acids with a molecular weight of 128,079. The use of a recent algorithm to predict membrane-protein interactions led to the unambiguous localization of the signal peptide and a transmembrane anchor alpha-helix at the C-terminus. At 50 positions amino acid differences were found between M41 and two Beaudette strains (M42-Salk and M42-Houghton). They are partly clustered in two regions of the protein. These two regions are candidates for neutralization epitopes of the protein.

COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1-defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 1 BRCA1 cells. By EJ5-and DR-green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 1 BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering druginduced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent.

In this report, we describe a novel lytic mechanism exploited by antimicrotubule drugs (AMDs) such as Taxol which are frequently used to treat multiple human cancers including breast and ovarian cancers. In cells lacking the G 1 -arresting capacity due to the defect in retinoblastoma or p53 gene function, AMDs trigger hyperploid progression and death. The hyperploid progression occurs via continued cellcycle progression without cell division. Blocking hyperploid progression through hydroxyurea or ectopically expressed p27 Kip1 , a G 1 -specific Cdk inhibitor, abrogates AMD cytotoxicity. Thus, AMDs induce lethality in G 1 -checkpointdefective cells by triggering hyperploid progression. The phenomenon is reminiscent of that observed previously with bub-1 yeast mutant. The potential significance of this finding lies in that hyperploid progression-mediated death may be exploited to develop a therapy with tumor-specificity at the genetic level. As a large fraction of human cancers are mutated in p53 gene, it may have a wide therapeutic applicability.

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