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R Bookstein

Publications and source records attributed to R Bookstein.

At least 37 records · Page 2Linked to original sources
PubMed1994

Comparative genomic hybridization, allelic imbalance, and fluorescence in situ hybridization on chromosome 8 in prostate cancer.

Due to problems with primary tumor cell culture, conventional cytogenetics has yielded little insightful information on chromosomal alterations in prostate cancer. The primary aim of this study was to define the ability of comparative genomic hybridization (CGH) to detect and map genetic deletions in prostate tumors. A secondary aim was to apply multiple assays to individual tumors as a means of deciphering the mechanisms of genetic alterations in prostate cancer. CGH results were compared with allelic imbalance measurements at 29 distinct loci on chromosome 8 in 18 specimens (17 malignant and 1 benign). CGH detected no changes in cases where all informative PCR/RFLP loci were retained and detected all p arm deletions consisting of at least two loci. We estimate that in this study, the smallest deletions detected by CGH were approximately 20-30 cM. Physical mapping of subchromosomal arm deletions by CGH correlated well with allelic imbalance mapping by PCR/RFLP: The data agreed at 88% of loci on 8p and 92% of loci on 8q. Fluorescence in situ hybridization (FISH) with multiple centromere probes and DNA content flow cytometry (FCM) also was performed on selected specimens. FISH revealed two cases of chromosome 8 aneusomy. In these two cases and three others, CGH showed simultaneous p arm deletion and q arm gain, suggesting isochromosome 8q formation. Together, these data suggested that, simple chromosomal aberrations were responsible for allelic losses on 8p and allelic gains on 8q in a significant number of prostate tumors. We also used CGH to examine relative DNA sequence copy number throughout the genome. Changes frequently associated with 8p loss include gains of 8q and losses of 13q, 16p, 16q, 17p, 17q, 20q, and Y. Cases with 8p loss exhibited five times the number of alterations as did cases without 8p loss.

Alleles↗
PubMed1994

The most promising surrogate endpoint biomarkers for screening candidate chemopreventive compounds for prostatic adenocarcinoma in short-term phase II clinical trials.

Surrogate endpoint biomarkers (SEBs) are needed in clinical chemoprevention trials to avoid the excessively long study periods and high costs associated with the use of cancer incidence reduction as an endpoint, particularly with relatively slow-growing tumors such as prostatic adenocarcinoma. SEBs should be directly associated with the evolution of neoplasia, and develop with high frequency in abnormal cells of susceptible individuals. If SEBs can be modified by a particular intervention regimen in short-term studies, the rationale for carrying out long-term studies may be strengthened. The consensus panel identified a small and manageable group of biomarkers measured in tissue or serum as the most promising in prostate cancer chemoprevention, including (1) prostate specific antigen (PSA); (2) morphometric markers, such as nuclear size and roundness; (3) proliferation markers, such as MIB-1 and PCNA; (4) nuclear DNA content (ploidy); (5) oncogene c-erbB-2 (HER-2/neu) expression; (6) angiogenesis; and (7) high-grade prostatic intraepithelial neoplasia (PIN). Information regarding many of these and other biomarkers is limited, calling for further investigation. Also, these factors, chosen chiefly for their proven or proposed utility as prognostic factors, may be less useful as SEBs. It was agreed that concurrent study of numerous markers rather than single markers allows comparison of their relative utility, including assessment of ease of quantitation and the sensitivity, specificity, and positive and negative predictive value.

Anticarcinogenic Agents↗
PubMed1994

Tumor suppressor genes in prostatic oncogenesis.

Mutations of tumor suppressor genes are critical genetic alterations occurring during the genesis and progression of human cancer, and consequently are candidates for use as surrogate endpoint biomarkers. The two most intensively studied suppressor genes, retinoblastoma (Rb) and p53, are mutated in approximately 20-50% of advanced-stage prostate cancers, but only rarely in early tumors. The precise DNA base changes, especially those affecting p53, may yield clues to relevant carcinogenic mechanisms. Increased expression of p53 in neoplastic cells, as detected by immunohistochemistry, may indicate mutation or a physiological response to DNA damage. Allelic losses of chromosome arms 8p and 16q are relatively common even in early prostate cancers. Quantitative measurement of allelic imbalance can be performed in preneoplastic or small neoplastic lesions, albeit with some technical challenge. The significance of whole-genome or regional allelic imbalance at various stages of prostatic oncogenesis has not been established.

Biomarkers, Tumor↗
PubMed1993

p53 is mutated in a subset of advanced-stage prostate cancers.

Inactivation of p53, a tumor suppressor gene, contributes to the genesis and/or progression of a substantial fraction of all human cancers, including > or = 50% of breast, lung, and colon carcinomas. Mutated p53 alleles typically contain missense single-base substitutions within exons 5-8 and encode abnormally stable p53 proteins that accumulate to high levels in tumor cell nuclei. To evaluate the frequency, type, and clinical significance of p53 mutation in human prostate cancer, archival tumor material from 150 prostate cancer patients was examined by immunohistochemistry (IHC) with anti-p53 antibodies. Abnormal nuclear p53 accumulation (IHC) was observed in 19 tumors (12.7%) and was strongly related to disease stage (23% of 69 stage III or IV tumors were IHC+ versus 4% of 74 stage 0-II tumors; P < 0.001, Fisher's exact test). The methods of polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing were used to identify mutations, predominantly missense single-base substitutions in exons 5, 7, or 8 in 9 of 14 IHC+ cases but in none of 20 IHC- cases; 5 of these mutations were G:C-->A:T transitions at CpG dinucleotides. These data indicate that mutated p53 alleles are quite uncommon in early prostate cancers but are found in 20-25% of advanced cancers, suggesting a role for p53 mutation in the progression of at least a subset of prostate cancers.

Amino Acid Sequence↗
PubMed1993

Recessive oncogenes.

Tumor-suppressor genes (antioncogenes or recessive oncogenes) are cancer genes that achieve their oncogenic effect by mutational inactivation of both normal alleles. By contrast, oncogenes are created from protooncogenes by mutations that lead to aberrant functional activation. Mutation of multiple suppressor genes and/or oncogenes probably is required for the genesis of most human neoplasms. Two well-characterized tumor-suppressor genes, the retinoblastoma gene (rb) on chromosome 13q and p53 on chromosome 17p, frequently are mutated in a broad range of human cancer types. Mutations of these genes have been documented in prostate carcinoma but appear to affect only a subset of cases. Nevertheless, as in other cancers, introduction of normal copies of rb or p53 suppresses the neoplastic properties of prostatic tumor cells carrying mutated alleles of the relevant gene. These results suggest that mutation of rb or p53 is involved in the genesis or progression of some prostate cancers. Frequent allelic losses of certain chromosome arms (especially 8p, 10p and q, and 16q) from prostatic cancer cells may indicate the involvement of novel suppressor genes located in these regions. Although the inactivation of suppressor genes appears to be a common genetic mechanism in human oncogenesis, the rates of mutation of particular genes vary widely with the type of cancer. It is unknown whether prostate cancers with or without mutation of rb, p53, or other suppressor loci differ biologically or prognostically; this is an area of active investigation. Fundamental understanding of the genetic lesions that occur during human oncogenesis has great potential for clinical application in diagnosis, prognosis, and therapy.

Alleles↗
PubMed1992

Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells.

Mutational inactivation of the retinoblastoma gene (RB) is an invariant feature of the childhood eye cancer retinoblastoma and of tumor cells derived therefrom. In a previous study, retrovirus-mediated transfer of wild-type RB into cultured retinoblastoma cells resulted in a marked enlargement and reduced growth rate of these cells, as well as loss of their tumorigenic properties in nude mice. It was therefore difficult to separate the proposed growth-suppressing and tumor-suppressing activities of RB protein. Here, we show that clones of RB-reconstituted retinoblastoma cells can be isolated that stably express apparently normal RB protein for at least 20 months of continuous culture. These clones were indistinguishable from nonreconstituted cells by multiple parameters including morphology, growth rate, and cell cycle distribution. Despite similar phenotypes in culture, clones with stable RB expression were uniformly nontumorigenic in nude mice, whereas those that lost such expression regained their tumorigenic properties. These results indicate that the tumorigenicity of these cells is entirely determined by the presence or absence of exogenous RB protein expression and that suppression of tumorigenicity is distinct from inhibition of cellular growth in culture.

Animals↗
PubMed1991

Retinoblastoma cell lines Y79, RB355 and WERI-Rb27 are genetically related.

Genesis of the childhood ocular tumor retinoblastoma results from the mutational inactivation of a single gene, RB, located on chromosome 13. Cultured cells or cell lines derived from retinoblastomas have been extensively studied for insight into mutational mechanisms of RB inactivation, functional properties of wild-type RB alleles, and pathways of retinal differentiation. Three such cell lines (Y79, RB355 and WERI-Rb27) were previously shown to have similar, heterozygous rearrangements of their RB genes, suggesting a common mutational mechanism affecting a specific region of the gene. This proposal was based on the premise that all three mutations occurred independently. By using molecular analyses of human genetic polymorphisms, we now show that these three cell lines are in fact genetically related, despite their different origins, morphologies, growth characteristics, and karyotypes. Interpretation of these and other published data suggest that both RB355 and WERI-Rb27 are probably sublines of Y79.

Blotting, Southern↗
PubMed1991

Molecular genetics of the retinoblastoma suppressor gene.

The retinoblastoma gene (RB) is the prototype of a class of genes, called tumor suppressor genes, for which loss-of-function mutations are oncogenic. Such genes would then normally function to suppress or prevent tumor formation. Classical genetic and cytogenetic studies of retinoblastoma, a rare childhood eye cancer, laid a fundamental groundwork for the molecular cloning of this gene. Surprisingly, mutations of RB are found not only in retinoblastomas but also in some osteosarcomas, soft-tissue sarcomas, and carcinomas of breast, lung, prostate or bladder, suggesting a broad role for RB in human oncogenesis. In support of this hypothesis, a wild-type copy of RB is able to suppress the neoplastic properties of several types of tumor cells with mutated endogenous RB alleles. The RB gene product, pp110RB, is a nuclear phosphoprotein with DNA binding activity. RB protein is cyclically phosphorylated and dephosphorylated during the cell division cycle, and may play a significant role in its regulation.

Amino Acid Sequence↗
PubMed1990

Genetic mechanisms of tumor suppression by the human p53 gene.

Mutations of the gene encoding p53, a 53-kilodalton cellular protein, are found frequently in human tumor cells, suggesting a crucial role for this gene in human oncogenesis. To model the stepwise mutation or loss of both p53 alleles during tumorigenesis, a human osteosarcoma cell line, Saos-2, was used that completely lacked endogenous p53. Single copies of exogenous p53 genes were then introduced by infecting cells with recombinant retroviruses containing either point-mutated or wild-type versions of the p53 cDNA sequence. Expression of wild-type p53 suppressed the neoplastic phenotype of Saos-2 cells, whereas expression of mutated p53 conferred a limited growth advantage to cells in the absence of wild-type p53. Wild-type p53 was phenotypically dominant to mutated p53 in a two-allele configuration. These results suggest that, as with the retinoblastoma gene, mutation of both alleles of the p53 gene is essential for its role in oncogenesis.

Alleles↗
PubMed1990

Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene.

Introduction of a normal retinoblastoma gene (RB) into retinoblastoma cells was previously shown to suppress several aspects of their neoplastic phenotype, including tumorigenicity in nude mice, thereby directly demonstrating a cancer suppression function of RB. To explore the possibility of a similar activity in a common adult tumor, RB expression was examined in three human prostate carcinoma cell lines. One of these, DU145, contained an abnormally small protein translated from an RB messenger RNA transcript that lacked 105 nucleotides encoded by exon 21. To assess the functional consequences of this mutation, normal RB expression was restored in DU145 cells by retrovirus-mediated gene transfer. Cells that maintained stable exogenous RB expression lost their ability to form tumors in nude mice, although their growth rate in culture was apparently unaltered. These results suggest that RB inactivation can play a significant role in the genesis of a common adult neoplasm and that restoration of normal RB-encoded protein in tumors could have clinical utility.

Animals↗
PubMed1990

Resolution of DNA linkage discrepancies through analysis of a VNTR locus in a family study of cystic fibrosis.

First-trimester prenatal diagnosis of a fetus at 25 per cent risk for cystic fibrosis (CF) was performed by indirect linkage analysis of polymorphic markers using Southern blotting and polymerase chain reaction (PCR) amplification. The results revealed discrepancies in the allelic patterns between the father and the affected child, thereby complicating the prediction of fetal outcome. Analysis of a highly polymorphic VNTR locus within the human retinoblastoma (RB) gene on chromosome 13 showed that the affected child and the fetus did not have the same biological father, and therefore the affected child could not be used to determine linkage of markers in the father of the fetus. The analysis of VNTR loci can be an effective method of resolving conflicting data during prenatal diagnosis of monogenic diseases.

Adult↗
PubMed1990

Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma.

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 of prostate 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 of five 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 (ii) 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 that RB can be inactivated in vivo by mutation of its promoter, (ii) confirm the existence of RB 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.

Autopsy↗
PubMed1990

Deletion of a splice donor site ablates expression of the following exon and produces an unphosphorylated RB protein unable to bind SV40 T antigen.

Studies of mutated retinoblastoma (RB) proteins in human tumor cells potentially reveal regions of the normal RB gene product that are required for its cancer suppression function. We here characterize a mutated RB protein of Mr 104,000 (p104) from a primary small-cell lung carcinoma. Unlike normal RB protein (pp110RB), p104 was unphosphorylated and unable to bind T antigen of SV40 both in vivo and in vitro. On the other hand, nuclear localization and DNA binding activity were preserved in the mutated protein. p104 was immunoprecipitable with four separate polyclonal antibodies recognizing different epitopes of the RB polypeptide, suggesting the presence of most exons in their correct reading frame. Following reverse transcription and in vitro amplification, RB mRNA from this tumor was shown to lack nucleotides encoded by exon 16. Analysis of genomic DNA from this tumor showed that exon 16 and its flanking splice donor and acceptor sequences were present and entirely normal; however, a 43-base pair (bp) region containing the splice donor site of intron 15 was deleted instead. Exon 15 was joined directly to exon 17 during mRNA processing via a cryptic splice donor site; exon 16 was presumably skipped because the preceding mutated intron was of insufficient length (less than 80 bp) for normal RB mRNA processing. These results demonstrate that loss of a single small exon disrupts several important biochemical properties of RB protein. In addition, sequence features of the 43-bp depletion suggest involvement of a novel deletional mechanism.

Antigens, Polyomavirus Transforming↗
PubMed1989

Structure of the human retinoblastoma gene.

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 greater than 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.

Base Sequence↗