Biomedical subjects

J Houldsworth

Publications and source records attributed to J Houldsworth.

At least 19 recordsLinked to original sources

PubMed1998

Chromosomal amplification is associated with cisplatin resistance of human male germ cell tumors.

Chemotherapy resistance of tumors is an important biological and clinical problem. Studies from many tumor types have indicated that resistance may be based on multiple genetic pathways. Human male germa cell tumors (GCTs) are an especially good model system to study the genetic basis of tumor sensitivity and resistance to chemotherapy. GCTs are exquisitely sensitive to treatment with DNA-damaging drugs such as cisplatin, rarely exhibit TP53 gene mutations, express normal p53 protein, and undergo p53-mediated apoptosis upon drug treatment. A small proportion of tumors (20-30% of metastatic lesions) escape the apoptotic response and result in treatment resistance. We have recently shown (J. Houldsworth, et al., Oncogene, 16: 2345-2359, 1998) that in a subset of such tumors, resistance is linked to TP53 gene mutations. In a further search for genetic mechanisms underlying resistance, we subjected a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemotherapy-resistant tumors to comparative genomic hybridization analysis to identify possible amplified regions (implying amplified/overexpressed genes) associated with resistance. With the exception of 12p11.2-12, high level amplification was not detected in any of the sensitive tumors. We have identified eight amplified regions (1q31-32, 2p23-24, 7q21, 7q31, 9q22, 9q32-34, 15q23-24, and 20q11.2-12) in five resistant tumors, which suggests that chromosomal and, hence, gene amplification may comprise a pathway to drug resistance. Identification of amplified/overexpressed genes at these sites may elucidate new genetic pathways of chemotherapy resistance in GCTs and possibly also in other tumors.

Chromosome Mapping↗

PubMed1998

Chromosomal and gene amplification in diffuse large B-cell lymphoma.

Chromosomal translocations leading to deregulation of specific oncogenes characterize approximately 50% of cases of diffuse large B-cell lymphomas (DLBL). To characterize additional genetic features that may be of value in delineating the clinical characteristics of DLBL, we studied a panel of 96 cases at diagnosis consecutively ascertained at the Memorial Sloan-Kettering Cancer Center (MSKCC) for incidence of gene amplification, a genetic abnormality previously shown to be associated with tumor progression and clinical outcome. A subset of 20 cases was subjected to comparative genomic hybridization (CGH) analysis, which identified nine sites of chromosomal amplification (1q21-23, 2p12-16, 8q24, 9q34, 12q12-14, 13q32, 16p12, 18q21-22, and 22q12). Candidate amplified genes mapped to these sites were selected for further analysis based on their known roles in lymphoid cell and lymphoma development, and/or history of amplification in tumors. Probes for six genes, which fulfilled these criteria, REL (2p12-16), MYC (8q24), BCL2 (18q21), GLI, CDK4, and MDM2 (12q13-14), were used in a quantitative Southern blotting analysis of the 96 DLBL DNAs. Each of these genes was amplified (four or more copies) with incidence ranging from 11% to 23%. This analysis is consistent with our previous finding that REL amplification is associated with extranodal presentation. In addition, BCL2 rearrangement and/or REL, MYC, BCL2, GLI, CDK4, and MDM2 amplification was associated with advanced stage disease. These data show, for the first time, that amplification of chromosomal regions and genes is a frequent phenomenon in DLBL and demonstrates their potential significance in lymphomagenesis.

Chromosome Mapping↗

PubMed1998

Human male germ cell tumor resistance to cisplatin is linked to TP53 gene mutation.

Male germ cell tumors (GCTs) are uniquely sensitive to cisplatin-based chemotherapy, with more than 90% of newly diagnosed cases cured. The underlying cause for resistance to treatment in 20-30% of metastatic lesions remains to be identified. Unlike other solid tumors, no mutations in the TP53 gene have been identified to date in random panels of GCT specimens, which could account for the exquisite sensitivity of these tumors to genotoxic insult. However, in a panel of resistant GCTs that did either not respond to cisplatin-based chemotherapy or subsequently relapsed and resulted in the death of the patient, we have now identified a subset of tumors to contain TP53 mutations within exons 6-9. A cell line derived from one of these tumors (228A) displayed the same TP53 mutation as the tumor specimen, expressed only mutant TP53 mRNA, and exhibited a relative resistance to cisplatin in vitro in comparison to a cell line (218A) derived from a responsive tumor with wild-type TP53. The resistant cell line displayed a much reduced apoptotic cell death and did not exhibit an induction of transcription of the p53-responsive genes WAF1 and MDM2 following cisplatin treatment, compared to that observed in the sensitive cell line. The levels of bax, an agonist of apoptosis, were found to be reduced in the resistant cell line. The simplest explanation for the resistance of this subset of GCTs that are resistant to cisplatin-based chemotherapy, is the inability of the cells to mount an apoptotic response following exposure due to a functionally inactivating mutation in the TP53 gene.

Antineoplastic Agents↗

PubMed1998

The cytogenetic theory of the pathogenesis of human adult male germ cell tumors. Review article.

Human male germ cell tumors (GCTs) represent a biological paradox because, in order to develop into a pluripotential tumor, a germ cell destined to a path of limited or no proliferation must acquire the potential for unlimited proliferation. In addition, it must acquire the ability to elicit embryonal differentiation patterns without the reciprocal inputs from fertilization and the imprinting-associated genomic changes which are a part of normal embryonal development. Although much speculated about, the genetic mechanisms underlying these properties of male GCTs remain enigmatic. Recent cytogenetic and molecular genetic analyses of these tumors are providing new insights and new testable hypotheses. Based on our recent work, we propose two such hypotheses. One relates to the mechanism of germ cell transformation and germ cell tumor development. We suggest that the invariable 12p amplification noted as early as in carcinoma in situ/intratubular germ cell neoplasia (CIS/ITGCN) lesions leads to deregulated overexpression of cyclin D2, a cell cycle G1/S checkpoint regulator with oncogeneic potential. Such overexpression reinitiates the cell cycle. We visualize this happening during the pachytene stage of meiosis through aberrant recombinational events which lead to 12p amplification. The other hypothesis relates to the origin of primary extragonadal GCTs. By comparing cytogenetic changes in primary mediastinal versus gonadal lesions, we propose that, in contrast to long-standing speculation that primary extra-gonadal tumors arise from embryonally misplaced primordial germ cells, these lesions arise from migration of transformed gonadal germ cells.

Adult↗

PubMed1997

Aberrant expression of cyclin D2 is an early event in human male germ cell tumorigenesis.

Human male germ cell tumors (GCTs) arise in the spermatocytic lineage, and subsets display embryonal-like differentiation. Virtually all GCTs exhibit multiple copies of the short arm of chromosome 12, even in carcinoma in situ/intratubular germ cell neoplasia, the earliest recognizable neoplastic lesion of germ cells. Among the candidate amplified genes mapped to 12p, expression of the cyclin D2 gene (CCND2) was deregulated in a panel of GCT cell lines, with the relative level of steady-state mRNA and protein inversely correlated with the pattern of differentiation characteristic of the cell line. GCT cell lines with a more differentiated phenotype, as indicated by an immunophenotypic analysis, displayed lower cyclin D2 expression with a concurrent increase in expression of the cell cycle inhibitor p21. In the GCT cell lines in which cyclin D2 was highly expressed, cyclin D2 was in complex with its expected catalytic partners (Cdk4 and Cdk6). Whereas no detectable cyclin D2 expression was evident in normal human germ cells, cyclin D2 was expressed in the abnormal germ cells of all carcinoma in situ/intratubular germ cell neoplasia lesions studied. In GCT specimens that displayed no evidence of differentiation (seminoma) or primitive differentiation (embryonal carcinoma), cyclin D2 expression was detected. However, in tumor specimens with certain patterns of differentiation (teratoma and yolk sac tumor), expression was down- or up-regulated depending on the pattern. Our data suggest that aberrant cyclin D2 expression is an early event in germ cell tumorigenesis.

Blotting, Northern↗

PubMed1996

REL proto-oncogene is frequently amplified in extranodal diffuse large cell lymphoma.

Comparative genomic hybridization (CGH) analysis of DNA extracted from a diffuse lymphoma with a large cell component (DLLC) that displayed double minute chromosomes upon conventional karyotypic analysis indicated overt amplification of DNA sequences derived from the 2p13-15 region. Southern blot analysis of this tumor DNA with a cDNA probe for the proto-oncogene REL, previously mapped to 2p14-15, indicated a greater than 35-fold amplification of REL. To determine the incidence of REL amplification and possible clinical or histologic association with DLLC, a panel of 111 tumor DNAs from DLLC specimens was screened for REL amplification by Southern blot analysis. A copy number of > or = 4 was noted in 26 cases (23%). Southern blot analysis of these 26 tumor DNAs with a cDNA probe for TGFA, mapped to 2p13, indicated lack of coamplification except in one case. Another member of the Rel/NF-kappa B family of transcriptional activators, RELA/p65 mapped to 11q13, was amplified in five cases as determined by Southern blot analysis using a cDNA probe. Nineteen of the 26 DLLC (73%) with REL amplification were primary extranodal lymphomas. As a group, the tumors with REL amplification demonstrated an increased frequency of chromosomal aberrations previously associated with tumor progression, suggesting an oncogenic effect of amplified REL in B-lymphoid cells that already contained a transforming genetic lesion. Thus, REL amplification is a frequent event in DLLC, and probably constitutes a progression-associated marker of primary extranodal lymphomas. This study shows the usefulness of the CGH technique in identifying chromosomal regions overrepresented in tumors that can point to amplified genes and may be correlated with clinical features of the disease.

Aneuploidy↗

PubMed1994

Gene amplification in non-Hodgkin's lymphoma.

Among 426 consecutively ascertained and karyotypically abnormal non-Hodgkin's lymphoma (NHL) tumours, cytological evidence for gene amplification in the form of homogeneously staining regions (HSRs) was encountered in nine cases of large cell diffuse lymphoma (LC-DL). The mean age of patients with HSRs was 62.9 years and four died within a year of diagnosis. To identify candidate gene(s) amplified in these tumours, we performed a Southern blot analysis of tumour DNA using probes for 23 known protooncogenes and the multidrug resistance gene, PGY1. Besides a two-fold amplification of the BCL2 gene in two cases, no evidence for overt amplification of any of the genes assayed was found. To confirm DNA amplification in these specimens we performed the DNA in-gel renaturation assay. Evidence for presence of amplified DNA fragments was obtained in four of seven specimens. These results suggest amplification of a novel gene(s). To our knowledge, this is the first formal study of gene amplification in a large consecutively ascertained series of fresh lymphoma biopsies.

Adult↗

PubMed1994

Allelic loss and somatic differentiation in human male germ cell tumors.

The complex but poorly understood human male germ cell tumors offer unusual opportunities for the genetic analysis of malignant transformation and embryonal differentiation in a pluripotential stem cell lineage. Histologically, these tumors are divided into two major subgroups, seminomas which are characterized by inability to express embryonal differentiation, and non-seminomas which are characterized by ability to express embryonal as well as extra-embryonal patterns of differentiation. To understand the role of genetic factors in the development of these tumors and the regulation of differentiation expressed by them, we carried out a detailed allelotype analysis by the loss of heterozygosity assay. This analysis revealed frequent deletions in known tumor suppressor genes (RB1, DCC, NME), a number of previously described sites of candidate tumor suppressor genes (3p, 9p, 9q, 10q, 11p, 11q and 17p), as well as several novel sites (2p, 3q, 5p, 12q, 18p and 20p). Our results also showed that well differentiated teratomas exhibit a significantly higher level of allelic loss compared to the less differentiated embryonal carcinomas. In addition, certain loci and genes exhibited frequent non-random deletion in teratomas (D3S32, D3S42, D5S12, D10S25, D11S12, RB1, TP53, NME1, NME2, D17S4, D18S6 and D20S6) and embryonal carcinomas (IFNB, D9S27). Among these loci, the NME genes were notable for a high degree of genetic loss (> 70%) in teratomas. These results suggested that nonrandom loss or inactivation of certain genes may be associated with tumor development and loss or inactivation of other genes may be associated with somatic differentiation.

Alleles↗

PubMed1994

Frequent allelic deletions and loss of expression characterize the DCC gene in male germ cell tumors.

The DCC tumor suppressor gene has been shown to be frequently deleted or its expression reduced or absent in colorectal, gastro-intestinal, pancreatic, prostatic, and breast carcinomas, and glioblastomas. By allelotype analysis using the DCC-flanking polymorphic marker D18S5 we have previously shown that allelic deletions at 18q21 occur in 40% of male germ cell tumors (Murty et al., 1994). In order to further understand the role of DCC gene in germ cell tumorigenesis, we evaluated deletions by loss of heterozygosity (LOH) and mRNA expression by RT-PCR in tumor tissues and cell lines. Analysis of 61 paired normal-tumor DNAs using the probes D18S5, JOSH 4.4 (a polymorphism within the DCC locus) and a (CA)n polymorphism in an intron of DCC revealed that 45% of GCTs had allelic deletions. In addition, two homozygous deletions were found in the DCC gene among 91 (61 used in the LOH analysis and an additional 30) tumor DNAs when screened with the cDNA probes (pDCC 1.65, pDCC 1.9 and pDCC 1.0). By RT-PCR analysis of four normal testes, nine GCT cell lines and 14 tumor tissues, DCC gene expression was detected in all four normal testes, while four (45%) GCT cell lines and one (7%) tumor specimen showed lack of expression. In addition, DCC expression was highly reduced in three (21%) tumor tissues. The high frequency of LOH at 18q21 was characteristic of seminomas as well as all subsets of non-seminomas in primary as well as metastatic states. Frequent allelic loss in all histologic subsets, homozygous deletions, and loss of expression of DCC suggest that suppression of this gene's function is an early event in GCT development.

Alleles↗

PubMed1994

Comparative genomic hybridization: an overview.

Comparative genomic hybridization (CGH) is a newly described molecular-cytogenetic assay that globally assays for chromosomal gains and losses in a genomic complement. In this assay, normal human metaphase chromosomes are competitively hybridized with two differentially labeled genomic DNAs (test and reference), which upon fluorescence microscopy, reveal the chromosomal locations of copy number changes in DNA sequences between the two complements. Application of CGH to DNAs extracted from fresh frozen specimens and cell lines of various tumor types has revealed a number of recurring chromosomal gains and losses that were undetected by traditional cytogenetic analysis. Few previously known sites were found to be in higher copy number, or lost by CGH, while many novel amplified regions were identified. These regions warrant further molecular genetic studies aimed at isolating the perturbed genes. Since CGH can also be performed on DNA extracted from formalin-fixed paraffin-embedded archived tumor specimens with few modifications, gains and losses of genetic material can be determined for specimens that would otherwise be unanalyzable. Prospective and retrospective application of CGH to tumor specimens would permit correlative studies to be performed, possibly identifying diagnostic and prognostic indicators of disease. CGH may also have a future role in detection and identification of chromosomal abnormalities in prenatal diagnosis and in dysmorphic anomalies.

Chromosome Mapping↗

PubMed1993

Molecular cytogenetic analysis of i(12p)-negative human male germ cell tumors.

The i(12p) chromosome has been shown to characterize more than 80% of male germ cell tumors (GCTs) and is an important diagnostic marker. Although recent cytogenetic analyses of GCTs have defined nonrandom chromosome abnormalities in these tumors, no attempt has so far been made to compare i(12p)-positive and -negative tumors in terms of their cytogenetic, histologic, and clinical features. During a 5-year period, we have ascertained 202 GCTs, of which 117 had clonally abnormal karyotypes. Among the latter, 91 had one or more copies of i(12p), whereas 26 lacked an i(12p). We report here the karyotypic analysis of these 26 i(12p)-negative GCTs. In this group, nonrandom sites of chromosomal rearrangements included 12p13 (9/26) and 1p11-q11 (5/26). Comparison of the cytogenetic features of i(12p)-negative tumors with i(12p)-positive tumors revealed the only significant difference to be rearrangements affecting 12p13 in the former (35%) as compared to their absence in the latter (3%). Hybridization of metaphase preparations of 9 i(12p)-negative tumors with a chromosome 12 painting probe and with a microdissected 12p painting probe revealed extra copies of chromosome 12 segments incorporated into marker chromosomes whose composition could not otherwise be resolved by banding analysis; all were shown to be derived from 12p. These data demonstrate that both i(12p)-negative and -positive groups are characterized by an increased copy number of 12p, which is consistent with a lack of significant clinical or biological difference between them. An increased 12p copy number thus is a specific aberration of significance to the development of germ cell tumors.

Chromosome Aberrations↗

PubMed1993

DNA amplification in human gastric carcinomas.

We recently identified a genomic domain at chromosome 10q26 that is highly amplified in the gastric carcinoma cell lines KATO III and SNU-16 and contains the BEK/K-sam gene, which encodes several growth factor receptors. A contiguous segment of 200 kb spanning this gene was amplified in five of 139 (3.6%) primary gastric carcinomas, all of them classified as poorly differentiated tumors. There was no amplification of this genomic region in a variety of other solid tumors. The overall frequency of gene amplification among the gastric carcinomas rose to 19.4% when MYC, ERBB2, and INT2 were included in the analysis, with significant association with advanced tumor stage. Amplification of various genomic regions in solid tumors may be more frequent than previously estimated.

Carcinoma↗

PubMed1992

Allelic deletions in the long arm of chromosome 12 identify sites of candidate tumor suppressor genes in male germ cell tumors.

Human male germ cell tumors (GCTs) result from malignant transformation of premeiotic or early meiotic germ cells and exhibit embryonal-like differentiation of the three germinal layers. The genetic basis of origin and expression of differentiated phenotypes by GCTs are poorly understood. Our recent cytogenetic analysis of a large series of GCTs has shown that two chromosome 12 abnormalities, an isochromosome for the short arm [i(12p)] and deletions in the long arm [del(12q)], characterize these tumors, which led us to suggest that the deletions represent loss of one or more candidate tumor suppressor genes whose products regulate the normal proliferation of the spermatogonial stem cells. We undertook a molecular mapping of the deletions by comparing germ-line and tumor genotypes of eight polymorphic loci in paired normal/tumor DNA samples from 45 GCT patients. Analysis of loss of constitutional heterozygosity at these loci revealed two regions of frequent loss (> 40%), one at 12q13 and the other at 12q22, identifying the sites of the postulated tumor suppressor genes. One tumor (no. 143A) exhibited a homozygous deletion of a region of 12q22, which included the MGF gene. The KIT and MGF genes have been shown to play key roles in embryonal and postnatal development of germ cells; therefore, we evaluated their expression by Northern blot analysis in a panel of three GCT cell lines and 24 fresh GCT biopsies. Deregulated expression of MGF and KIT, which was discordant between seminomatous and nonseminomatous lesions, was observed.

Alleles↗

PubMed1992

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.

Blotting, Southern↗

PubMed1992

Analysis of BCL2 and MYC expression in non-Hodgkin's lymphomas by in situ hybridization: correlation with chromosome translocations.

We have used an in situ hybridization method for analysis of expression of BCL2 and MYC on cytospun preparations of normal and malignant lymphoid cell lines and tissue sections of normal and malignant lymph nodes. The probes comprised 50-mer antisense oligonucleotides starting at the ATG codons of exon 3 of BCL2 and exon 2 of MYC. We studied the expression of these two genes in frozen tissue sections of biopsy specimens derived from normal and hyperplastic lymph nodes, B-cell lymphomas carrying the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, and T-cell lymphomas with clonal chromosome abnormalities. While all proliferating cells expressed both genes, BCL2 expression was increased two- to threefold in follicular lymphomas with t(14;18) and MYC expression was increased two- to four-fold in high-grade lymphomas with t(8;14). These results are consistent with previous data on deregulated expression of these genes obtained from study of lymphoma cell lines carrying the relevant translocations.

Base Sequence↗

PubMed1991

Extraction of DNA from paraffin blocks for Southern blot analysis.

Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.

Autoradiography↗

PubMed1991

Fanconi anemia: a pleotropic mutation with multiple cellular and developmental abnormalities.

Fanconi anemia (FA), an autosomal recessive disorder of children, is characterized by congenital or childhood aplastic anemia, multiple developmental anomalies, increased incidence of myeloid leukemia, increased spontaneous chromosome breakage, and cellular and chromosomal hypersensitivity to DNA bifunctional crosslinking and alkylating agents. Attempts to understand the biochemical basis of the disorder over the past two and a half decades have resulted in a number of descriptive studies pertaining to cytogenetic and biochemical abnormalities, especially of DNA repair proficiency following treatment with DNA bifunctional crosslinking agents. More recent approaches such as DNA transfection offer the potential for isolation and molecular characterization of the gene. The FA gene is postulated to belong to the family of genes that regulate the development of hematopoietic and other cell types.

Abnormalities, Multiple↗

PubMed1991

The response of ataxia-telangiectasia lymphoblastoid cells to neutron irradiation.

The response of control and ataxia-telangiectasia (A-T) cells to increasing doses of high-linear-energy-transfer (LET) ionizing radiation (neutrons) was compared. Ataxia-telangiectasia cells were markedly more sensitive to neutron irradiation than were control cells. The D0 value for the two A-T cell lines was 0.4 Gy while the value for controls was approximately 1.4 Gy. Fast neutrons were considerably more effective than gamma rays in inducing cell death in both cell types, but the sensitivity factor remained approximately the same as with gamma rays. A minimal depression of DNA synthesis was observed in ataxia-telangiectasia cells after neutron irradiation, similar to that reported previously after gamma irradiation. The extent of inhibition was not significantly greater in control cells, contrary to that seen with gamma rays. In time-course experiments a significant difference in degree of inhibition of DNA synthesis was observed between the cell types. Low doses of fast neutrons induced a G2-phase delay in both cell types, but the degree and extent of this delay was greater in ataxia-telangiectasia cells as observed previously with low-LET radiation.

Ataxia Telangiectasia↗