Biomedical subjects

R Bookstein

Publications and source records attributed to R Bookstein.

50 records · Page 3Linked to original sources

PubMed1989

Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line.

Mutational inactivation of the retinoblastoma (RB) gene is considered a fundamental event in the formation of several types of human cancer. A substantial proportion of RB gene mutations are partial or complete deletions that extend an unknown distance beyond one or both ends of the gene. To provide a framework for measuring the extent of these deletions, we have constructed a long-range restriction map of SfiI sites spanning 850 kilobases around the RB gene. This map was applied in a molecular analysis of RB gene deletion in breast cancer cell line MB468. A previous study of this cell line demonstrated deletion of the entire RB gene except for exons 1 and 2 (E. Y.-H. P. Lee, H. To, J.-Y. Shew, R. Bookstein, P. Scully, and W.-H. Lee, Science 241:218-221, 1988). Genomic clones containing the deletion junction were isolated from a library made from MB468 DNA. A probe obtained from the far side of the deletion junction was used to localize and clone the unknown 3' endpoint, demonstrating that the chromosomal mutation in this case was a simple deletion spanning 200 kilobases. Sequence analysis of the deletion junction indicated a conservative deletion with no loss or gain of nucleotides. The deletion endpoints had no sequence homology to each other or to any repetitive sequence family, such as Alu, so the recombination event was illegitimate. Structural analysis of this and other RB gene deletions is important for understanding molecular mechanisms of recessive oncogenesis.

Base Sequence↗

PubMed1988

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells.

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.

Cell Division↗

PubMed1988

Inactivation of the retinoblastoma susceptibility gene in human breast cancers.

Mutational inactivation of the retinoblastoma susceptibility (RB) gene, a recessive cancer gene, has been implicated in the genesis of retinoblastoma and certain other human neoplasms. This gene is now shown to be inactivated in two of nine human breast cancer cell lines examined. The RB gene of one cell line had a homozygous internal duplication of a 5-kilobase region containing exons 5 and 6. The RB messenger RNA transcript was correspondingly lengthened, and its translation was probably terminated prematurely due to a shifted reading frame. The other cell line had a homozygous deletion of the RB gene that removed the entire gene beyond exon 2. The RB gene product, pp110RB, was not detectable in either cell line by immuno-precipitation with specific antibodies. These findings are significant in relation to proposed genetic mechanisms of breast cancer formation.

Breast Neoplasms↗

PubMed1988

Studies on the human retinoblastoma susceptibility gene.

The retinoblastoma susceptibility (RB) gene is unique among other cloned cancer genes because its causal role in a human cancer, retinoblastoma, was established by classical genetic methods before its isolation. Earlier hypotheses and experimental data suggested that inactivation of a gene in chromosome band 13q14 resulted in retinoblastoma formation. A gene in this region was identified as the RB gene on the basis of mutations found specifically in retinoblastoma tumors; however, its proposed biological activity in suppressing neoplasia has yet to be demonstrated. The RB gene product was identified as a nuclear phosphoprotein of 110 kD associated with DNA binding activity, suggesting that the RB protein may regulate other genes. Probes for the RB gene and gene product will be useful for genetic diagnosis of retinoblastoma susceptibility in affected families; for direct detection of mutant RB alleles; and, potentially, for genetic diagnosis of susceptibility to osteosarcoma and other tumors tentatively linked to RB-gene dysfunction. Continued study of the RB gene should yield further insight into mechanisms of oncogenesis, development, and gene regulation.

Chromosome Mapping↗

PubMed1988

Human esterase D gene: complete cDNA sequence, genomic structure, and application in the genetic diagnosis of human retinoblastoma.

The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.

Amino Acid Sequence↗

PubMed1988

A case of synovial sarcoma with abnormal expression of the human retinoblastoma susceptibility gene.

Abnormal or absent expression of the human retinoblastoma susceptibility (RB) gene has been implicated in the genesis of retinoblastoma. In addition to a 90% chance of developing retinoblastoma, patients inheriting a mutant RB gene have a high incidence of second nonocular malignancies, suggesting a role for this gene in other tumors as well. This report describes a patient without a previous history of retinoblastoma who developed metastatic synovial sarcoma. Analysis of RNA from this tumor revealed the presence of an abnormally long RB gene transcript in addition to an RB transcript of normal length. On the basis of findings in retinoblastoma, we propose that alteration of RB gene expression was significantly related to the formation of this patient's synovial sarcoma.

Adult↗

PubMed1988

Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79.

Formation of retinoblastoma, a cancer arising in the retinas of young children, is determined by mutational inactivation of an autosomal gene (RB), which has been molecularly cloned. Whereas all normal tissues and many tumor cells express an RB mRNA of 4.7 kilobases, six of six retinoblastomas were previously found either to lack RB gene expression or to have RB transcripts of abnormal (reduced) length. To further characterize the latter type of mutation, we chose to examine retinoblastoma cell line Y79, which expressed a shortened RB mRNA of about 4.0 kilobases. RB cDNA clones isolated from a library constructed with Y79 mRNA demonstrated an internal loss of 470 nucleotides near the 5' end, which corresponded to a deletion of exons 2-6. Genomic clones containing the deletion junction were isolated from a library made with Y79 DNA, which allowed precise localization and sequencing of deletion endpoints in introns 1 and 6. These regions had no apparent homology to each other or to the Alu family of repetitive sequences, implying that the deletion must have occurred by a mechanism other than recombination of homologous sequences. Deletion of exons 2-6 would interrupt the open reading frame in RB mRNA and would result in premature termination of translation. Since no normal RB protein was detected by immunoprecipitation with specific antibody, the other, apparently normal RB allele in Y79 cells was necessarily inactivated by a different mutation.

Base Sequence↗

PubMed1988

Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families.

Cell Line↗

PubMed1988

An analysis of serum enzyme changes and clinical biochemical abnormalities of the anemia in Olympic runners.

In order to assess the changes in the clinical biochemistry of runner's anemia and its evolution during a prolonged period of high-intensity training, 11 male international class distance runners (mean time for 1 mile 4 min, 2.5 sec) were followed over a 10-month period prior to the 1984 U.S. Olympic Trials. Mean values of hemoglobin, hematocrit, and mean corpuscular hemoglobin (MCH) decreased modestly over the period of study. Means of haptoglobin, iron, and total iron binding capacity (TIBC) remained roughly constant. Percentage of saturation of TIBC by iron (% sat) averaged 30% or less in 5 of 11 runners, suggesting mild iron deficiency. Most measured haptoglobin levels were below normal range throughout the study period. The cause of runner's anemia has been demonstrated to be multifactorial, including disordered iron metabolism, iron deficiency, and hemolysis. Other studies have shown absent bone marrow iron in male athletes, secondary to hematuria, ischemia of the intestinal mucosa with bleeding, and iron losses due to heavy perspiring. Cardiorespiratory fitness, evaluated through repetitive treadmill testing, was not adversely affected in our athletes. Total creatine kinase (CK) increased significantly after a training session, while the MB fraction of CK never exceeded 3%. Total lactate dehydrogenase (LD) also rose after exercise, but the fractions represented by isozymes 1-5 were unaltered; specifically, there was no change in the LD-1/LD-2 ratio. Enzyme elevations were thus derived from skeletal muscle and not from heart.

Anemia↗

PubMed1987

Human retinoblastoma susceptibility gene: cloning, identification, and sequence.

Recent evidence indicates the existence of a genetic locus in chromosome region 13q14 that confers susceptibility to retinoblastoma, a cancer of the eye in children. A gene encoding a messenger RNA (mRNA) of 4.6 kilobases (kb), located in the proximity of esterase D, was identified as the retinoblastoma susceptibility (RB) gene on the basis of chromosomal location, homozygous deletion, and tumor-specific alterations in expression. Transcription of this gene was abnormal in six of six retinoblastomas examined: in two tumors, RB mRNA was not detectable, while four others expressed variable quantities of RB mRNA with decreased molecular size of about 4.0 kb. In contrast, full-length RB mRNA was present in human fetal retina and placenta, and in other tumors such as neuroblastoma and medulloblastoma. DNA from retinoblastoma cells had a homozygous gene deletion in one case and hemizygous deletion in another case, while the remainder were not grossly different from normal human control DNA. The gene contains at least 12 exons distributed in a region of over 100 kb. Sequence analysis of complementary DNA clones yielded a single long open reading frame that could encode a hypothetical protein of 816 amino acids. A computer-assisted search of a protein sequence database revealed no closely related proteins. Features of the predicted amino acid sequence include potential metal-binding domains similar to those found in nucleic acid-binding proteins. These results provide a framework for further study of recessive genetic mechanisms in human cancers.

Amino Acid Sequence↗

PubMed1987

A null allele of esterase D is a marker for genetic events in retinoblastoma formation.

The development of homozygosity or hemizygosity in the 13q14 region by deletion, mitotic recombination, or chromosomal loss has been interpreted as a primary event in retinoblastoma. This finding is consistent with the hypothesis that inactivation of both alleles of a gene located at 13q14.11 is required for tumorigenesis. Observations reported by Benedict and colleagues in one case of bilateral retinoblastoma, LA-RB 69, provided early evidence in favor of this hypothesis. By examining levels of esterase D, an enzyme also mapping to 13q14.11, it was previously inferred that one chromosome 13 was lost. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, we have found that low but detectable quantities of esterase D protein and enzymatic activity are present in tumor cells from LA-RB 69; fibroblasts from this patient contain two copies of the esterase D gene, indicated by heterozygosity at an Apa I polymorphic site within this gene; and tumor cells from the same patient are homozygous at this site, indicating loss and reduplication of the esterase D locus. These results demonstrate that one of the two esterase D alleles in this patient acted as a "null" or silent allele--that is, was present in the genome with markedly decreased protein expression. This mutant allele acted as a marker for tumor-associated loss of chromosome 13 heterozygosity, in concordance with previous proposals.

Alleles↗

PubMed1987

Changes in serum enzymes, lactate, and haptoglobin following acute physical stress in international-class athletes.

Skeletal muscle is rich in creatine kinase (CK), lactate dehydrogenase (LD), and other enzymes. Many reports describe changes in serum CK and LD following exercise. In our study, 11 male international-class medium-distance runners were followed over a 10-month period prior to the 1984 US Olympic Trials. Cardiorespiratory fitness, evaluated through repetitive treadmill testing, was unchanged in our athletes. Total CK increased significantly during the course of training, and the CK-MB activity was higher than that of sedentary individuals; CK-MB never rose to more than 3% of the total CK. Total LD also rose following acute exercise; however, the proportions of the five isoenzymes were unaltered. There was no change in the LD-1/LD-2 ratio from normal. The origin of the increased serum enzymes was believed to be primarily skeletal muscle. A decrease of serum haptoglobin following acute stress was attributed to intravascular hemolysis and binding of hemoglobin. As expected, serum lactate was dramatically increased immediately postexercise.

Adult↗

PubMedDate not supplied

The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity.

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.

Base Sequence↗

PubMedDate not supplied

Characteristics of two new retinoblastoma cell lines: WERI-Rb24 and WERI-Rb27.

From 49 eyes enucleated for retinoblastoma, two new cell lines, WERI-Rb24 (W-24) and WERI-Rb27 (W-27), were established in long-term culture (greater than 5 years). The W-24 cell line was derived from a 22-month-old boy with sporadic retinoblastoma; the W-27 cell line was derived from a 24-month-old boy with bilateral retinoblastoma. Both cell lines show abnormal mRNA transcripts corresponding to the retinoblastoma gene. At the DNA level, one retinoblastoma allele was not present in the W-24 cell line. In the W-27 cell line a mutation was identified in one allele, while the other appears grossly normal. Since no normal retinoblastoma mRNA can be detected in either cell line, a subtle mutation must occur on the grossly normal allele. Comparison of DNA in W-27 lymphocytes and tumor cells with DNA probe p6NR-0.5 indicates that the observed mutation occurred somatically. The application of these two new retinoblastoma cell lines to the characterization of defects in the retinoblastoma gene and to gene replacement therapy in retinoblastoma is discussed.

Alleles↗