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W F Benedict

Publications and source records attributed to W F Benedict.

At least 91 records · Page 5Linked to original sources
PubMed1987

Structural evidence for the authenticity of the human retinoblastoma gene.

The retinoblastoma (Rb) gene is the prototype for a class of recessive human cancer genes in which loss of activity of both normal alleles is thought to be associated with tumorigenesis. Sixteen of 40 retinoblastomas examined with a complementary DNA probe shown to be the Rb gene had identifiable structural changes of the Rb gene including in some cases homozygous internal deletions with corresponding truncated transcripts. An osteosarcoma also had a homozygous internal deletion with a truncated transcript. In addition, possible hot spots for deletion were identified within the Rb genomic locus. Among those tumors with no identifiable structural changes there was either absence of an Rb transcript or abnormal expression of the Rb transcript. Comparison of the structural changes in the tumor cells and fibroblasts of certain patients provided support for Knudson's two-hit hypothesis for the development of retinoblastoma at the molecular level. The ability to detect germline structural deletions in fibroblasts from some patients with bilateral retinoblastoma also indicates that the isolated gene is useful for diagnostic purposes.

Alleles↗
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↗
PubMed1986

Prediction of familial predisposition to retinoblastoma.

Retinoblastoma is a childhood cancer, predisposition to which is inherited as an autosomal dominant trait. We used restriction-fragment-length and isozymic alleles of loci on chromosome 13 in five families predisposed to retinoblastoma, to provide identification before illness of persons likely to have tumors. The likelihood of disease was predicted in two cases, and freedom from disease in three. The calculated predictive accuracy was greater than 94 percent in cases with informative loci flanking the retinoblastoma (RB1) locus, and our prediction has been fulfilled in each such instance. A case that was informative at several loci indicated the occurrence of meiotic recombination, and accurate prediction was based on data obtained with DNA markers and isozymic forms of esterase D. The calculated predictive accuracy in another case, which was informative only for loci distal to the retinoblastoma locus, was about 70 percent. This patient was expected to acquire the disease but had not done so at the age of one year, illustrating the need for more markers that are also more informative and genetically closer to the retinoblastoma locus. These studies provide the basis for prenatal and postnatal prediction of susceptibility to inherited cancer using arbitrary recombinant DNA markers. Such predictions should make genetic counseling for familial retinoblastoma more accurate and lead to earlier tumor detection and more effective therapy.

Alleles↗
PubMed1986

Purification, biochemical characterization, and biological function of human esterase D.

Human esterase D (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxymethylcellulose, phenyl-Sepharose, chromatofocusing, and hydroxylapatite chromatographies resulted in a 10,000-fold purification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 X 10(-6) M using 4-methylumbelliferyl acetate as substrate. The enzymatic activity was inhibited by p-chloromercuribenzoate and HgCl2, suggesting an important role of SH group(s) in enzyme function. Specific rabbit polyclonal and mouse monoclonal antibodies against esterase D were prepared and recognized either denatured or native human esterase D protein. Moreover, the polyclonal antibodies immunoprecipitated a polypeptide with a molecular mass of about 33-34 kDa from various cell lines of different mammalian species, indicating that the esterase D protein is highly conserved. The highest levels of this enzyme were found in liver and kidney. Furthermore, the expression of esterase D was enhanced 3-fold in a promonocytic cell line treated with phenobarbital but not with phorbol myristate acetate, suggesting that esterase D may have a role in detoxification. The availability of the homogeneous protein and its specific antibodies allows for cloning of the esterase D gene and facilitates studies of retinoblastomas.

Antibodies↗
PubMed1986

Implication of chromosome 11 in the suppression of neoplastic expression in human cell hybrids.

Cytogenetic analyses of intraspecies human HeLa X fibroblast hybrid cell populations have provided tentative evidence for the correlation of loss of a single copy of chromosomes 11 and 14 with reexpression of tumorigenicity. In this study paired combinations of nontumorigenic and tumorigenic segregant HeLa X fibroblast hybrid cells from two independent fusion events were examined for the presence or absence of normal chromosomes 11 and 14. In human hybrid cell lines the parental origin of chromosomes can be distinguished on the basis of restriction fragment length polymorphisms. Genes for c-Ha-ras, insulin, and apolipoprotein A-1 on chromosome 11 and a polymorphic locus AW101 on chromosome 14 were used as Southern hybridization probes. Analysis of DNA from the parental fibroblast and HeLa cell lines and their nontumorigenic and tumorigenic hybrids showed the loss of a fibroblast chromosome 11 in four of the tumorigenic segregants and a HeLa chromosome 11 in a fifth hybrid cell line. This latter segregant has, interestingly, also lost a copy of chromosome 14 of fibroblast origin. There was no obvious correlation of loss of a copy of normal chromosome 14 and reexpression of tumorigenicity in any of the other hybrid cell populations. Our conclusion from these observations is that gene(s) that map to normal chromosome 11 might be involved in control of tumorigenic expression in these human hybrid cells.

Alleles↗
PubMed1985

Oncogenic transformation of C3H/10T1/2 Cl 8 mouse embryo fibroblasts by inhibitors of nucleotide metabolism.

Morphological or oncogenic transformation of mouse embryo, C3H/10T1/2 Cl 8 fibroblasts was induced by methotrexate, 5-fluorouracil and 5-fluorodeoxyuridine. It is known that these compounds cause inhibition of thymidylate synthetase and, hence, depletion of deoxythymidine triphosphate (dTTP) and an increased ratio of deoxycytidine triphosphate (dCTP) to dTTP in the deoxyribonucleotide pools that are used for DNA synthesis in mammalian cells. This ratio is, in effect, increased by treating mammalian cells with arabinosyl cytosine and 5-azacytidine, which are converted into analogs of dCTP in mammalian cells and also induce oncogenic transformation of C3H/10T1/2 cells. By contrast, trifluorothymidine, 5-bromodeoxyuridine and 5-iododeoxyuridine, which are analogs of thymidine that in effect reduce the dCTP:dTTP ratio, did not induce oncogenic transformation. Moreover, thymidine was selectively lethal to tumorigenic C3H/10T1/2 cells and inhibited oncogenic transformation in cells treated with 5-fluorodeoxyuridine. These observations suggest that treatments that effectively increase the dCTP:dTTP ratio in mammalian cells facilitate oncogenic transformation of C3H/10T1/2 cells, whereas treatments that have the effect of decreasing this ratio inhibit transformation. However, dCyd did not induce oncogenic transformation of C3H/10T1/2 cells, although it has been shown to increase the dCTP:dTTP ratio in mammalian cells. Thus, increasing this ratio may not be sufficient to cause the transformation.

Animals↗
PubMed1985

Asbestos-associated chromosomal changes in human mesothelial cells.

Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of greater than 50 divisions beyond the culture life span (30 doublings) of the control cells.

Asbestos↗
PubMed1985

Retinoblastoma gene: a human cancer recessive (regulatory?) susceptibility gene.

The Rb susceptibility gene located in chromosomal region 13q14 apparently represents one of a class of recessive human cancer genes that can be contrasted to known oncogenes that function following amplification, aberrant insertion, or base substitutions at key sites within their gene. Loss of function of both Rb alleles appears to characterize the mechanism by which the Rb gene produces tumors, which may also include the second primary malignancies seen with high frequency in patients with the hereditary forms of retinoblastoma. Since the Rb gene may have a "suppressor" or "regulatory" function (which could include the regulation of an oncogene), cloning of such a human cancer susceptibility gene would seem highly worthwhile. Our laboratory, as well as several others, is therefore currently attempting to clone this gene.

Chromosomes, Human, 13-15↗
PubMed1984

Imaging of human tumor xenografts in nude mice with radiolabeled monoclonal antibodies. Limitations of specificity due to nonspecific uptake of antibody.

To determine if in vivo binding specificities of monoclonal antibodies to tumor nodules would reflect in vitro antibody specificities as determined by radioimmunoassay, two monoclonal antibodies were selected for imaging of human tumor xenografts in nude mice. By in vitro radioimmunoassay, antibody 436 binds to the M20 melanoma cell line, but not to the P3 carcinoma cell line; antibody 44 binds to P3 but not to M20. Both antibodies are IgG1 isotypes. Nude mice bearing an M20 melanoma in one flank and a P3 carcinoma in the other were injected intravenously with 5 to 25 micrograms of each antibody labeled with either I-125 or I-131; in separate animals the labels were reversed. Animals were imaged daily with a scintillation camera equipped with a pinhole collimator. On day 6 the animals were sacrificed, and binding of the antibodies to the tumors and normal tissue were compared. Antibody 436 had up to a two-fold binding advantage in vivo for the melanoma, whereas antibody 44 had up to a two-fold binding advantage for the carcinoma, thus confirming the in vitro specificities of each. However, imaging on day 4 and computer analysis of percent radioactivity in the tumors showed that tumor images were related directly to tumor size and relatively uninfluenced by antibody specificity. Thus, even though antibody specificity can be demonstrated by differential tissue counting, imaging of tumor deposits appears to involve a nonspecific phenomenon that is largely dependent upon tumor weight.

Animals↗
PubMed1984

Retinoblastoma: clues to human oncogenesis.

The retinoblastoma gene can be considered a model for a class of recessive human cancer genes that have a "suppressor" or "regulatory" function. The loss or inactivation of both alleles of this gene appears to be a primary mechanism in the development of retinoblastoma. Such a mechanism is in direct contrast to that of putative human oncogenes which are thought to induce tumorigenesis following activation or alteration. The high incidence of second primary tumors among patients who inherit one inactive retinoblastoma allele also suggests that this cancer gene plays a key role in the etiology of several other primary malignancies. Finally, the observation that extra nonrandom copies of specific chromosomal regions occur in some of these tumors provides circumstantial evidence that an "expressor" gene (possibly an oncogene) may be involved in retinoblastoma development.

Adenocarcinoma↗
PubMed1984

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke.

Using defined cigarette smoke exposure conditions, BC3F1/Cum mice were exposed nose-only to two different types of whole cigarette smoke on a daily basis for 1 week and up to 46 weeks. The number of sister-chromatid exchanges (SCEs) per metaphase was determined in bone-marrow cells. Studies were scheduled so that all cytogenetic observations were made 2-3 days after the last smoke exposure. Exposure to either type of smoke on a daily basis for 1 week or up to 46 weeks resulted in a 2-fold increase in SCEs over sham-exposed control mice. In animals exposed either chronically or for 1 week to either type of smoke, the increase in SCEs persisted for at least 1 week after cessation of smoke exposure. This is the first demonstration of the induction of SCEs in laboratory animals that have been exposed to cigarette smoke in vivo.

Animals↗
PubMed1984

Establishment and characterization of a human T-cell leukemia line (LALW-2) in nude mice.

Lymphoblasts from the peripheral blood of a 10-year-old boy who was treated for T-cell acute lymphoblastic leukemia were heterotransplanted intraocularly into nude mice. The resultant tumors and their metastases were serially passaged both intraocularly and subcutaneously in the mice. The mouse tumors closely resembled morphologically and cytochemically a human T-cell lymphoma. The primary cells, as well as cells from subsequent in vivo passages, were predominantly of a suppressor T-cell phenotype (OKT8-positive). No viral products were identified. Chromosome analysis revealed a near-diploid karyotype with a translocation between chromosomes 11 and 14. The tumorigenicity, morphology, cytochemistry, immunologic phenotype, and karyotypic pattern of the cells remained constant through six serial in vivo passages over a period of 10 months. This is the first report of direct heterotransplantation and long-term in vivo maintenance of primary human T-cells in immunologically unmanipulated nude mice.

Animals↗
PubMed1984

Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: chromosome dosage dependency.

The tumorigenic capacity of hybrids formed by fusion of the highly tumorigenic HT1080 human fibrosarcoma cell line with nontumorigenic normal fibroblasts was examined. The HT1080 also contains an activated N-ras oncogene. Near-tetraploid hybrids which contained an approximately complete chromosomal complement from both parental cells were nontumorigenic when 1 X 10(7) cells were injected s.c. into athymic (nude) mice, whereas the parental HT1080 cells produced tumors in 100% of the animals with no latency period following injection of 2 X 10(6) cells. Tumorigenic variants were obtained from these hybrids which had lost only a few chromosomes compared to cells from the nontumorigenic mass cultures. In addition, several near-hexaploid hybrids were obtained which contained approximately a double chromosomal complement from the HT1080 parental line and a single chromosomal complement from the normal fibroblasts. All of these near-hexaploid hybrids produce tumors in 100% of nude mice with no latency period. Our results indicate that tumorigenicity of these particular human malignant cells of mesenchymal origin can be suppressed when fused with normal diploid fibroblasts. In addition, the results suggest that tumorigenicity in this system is chromosomal dosage dependent, since a diploid chromosomal complement from normal fibroblasts is capable of suppressing the tumorigenicity of a near-diploid but not a near-tetraploid chromosomal complement from the tumorigenic HT1080 parent. Finally, the loss of chromosome 1 (the chromosome to which the N-ras oncogene has been assigned) as well as chromosome 4 was correlated with the reappearance of tumorigenicity in the rare variant populations from otherwise nontumorigenic near-tetraploid hybrid cultures. Our results also suggest the possibility that tumorigenicity in these hybrids may be a gene dosage effect involving the number of activated N-ras genes in the hybrids compared to the gene(s) controlling the suppression of the activated N-ras genes.

Cell Fusion↗
PubMed1983

Patient with 13 chromosome deletion: evidence that the retinoblastoma gene is a recessive cancer gene.

Although a constitutional chromosomal deletion including 13q14 has been found to date in all retinoblastoma patients whose esterase D activity is 50 percent of normal, one female patient has been found who has 50 percent esterase D activity in all normal cells examined but no deletion of 13q14 at the 550-band level. Therefore, she has the smallest constitutional chromosomal deletion within 13q14 that is associated with susceptibility to retinoblastoma. Two stem lines were identified in a retinoblastoma from this patient, and each one had a missing 13 chromosome. No detectable esterase D activity was found in the tumor, indicating that the normal nondeleted 13 chromosome was lost in both stem lines. Thus the data from this patient not only show that there is a total loss of genetic information at the location of the retinoblastoma gene within the tumor, but also imply that recessive genes may play an important role in the development of certain human tumors including retinoblastoma.

Child, Preschool↗
PubMed1983

Gene for hereditary retinoblastoma assigned to human chromosome 13 by linkage to esterase D.

Evaluation of three families with hereditary retinoblastoma demonstrates close linkage of the gene for this tumor with the genetic locus for esterase D. These results assign the gene for the hereditary form of retinoblastoma to band q14 on chromosome 13, the same region which is affected in the chromosome deletion form of this eye tumor, and therefore suggest a common underlying mechanism in the pathogenesis of these two forms of retinoblastoma.

Chromosome Deletion↗