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J Lubinski

Publications and source records attributed to J Lubinski.

At least 19 recordsLinked to original sources
PubMed2001

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes.

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Novel mechanism of antibody-independent complement neutralization of herpes simplex virus type 1.

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.

Adult↗
PubMed2000

Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer.

The aim of this study was to evaluate the significance of pedigree/clinical data, immunohistochemistry (IHC) and microsatellite instability (MI) analyses in the reduction of costs of constitutional hMLH1 and hMSH2 gene mutation diagnosis in patients with colorectal cancers (CRC). Pedigree/clinical data were evaluated on a series of 168 patients with CRC, including 43 consecutive sporadic late-onset and 25 consecutive, definitive or suspected hereditary non-polyposis colorectal cancer (HNPCC) cases, examined by IHC and MI analyses. In the latter group, 6/25 (24%) constitutional mutations were found. We detected no germline mutations in the sporadic late-onset patients. The lowest costs (880 Euro/mutation detected) were achieved by performing pedigree/clinical data (for exclusion of late-onset sporadic CRC) in conjuction with IHC only. In this model 1/6 (17%) mutations was missed. Additional preselection by IHC and MI analyses before sequencing was required to detect all mutations. In this approach, which seems to be the most effective in the search for hMLH1 and hMSH2 gene mutation, the cost was 1767 euro/mutation detected.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Animals↗
PubMed1999

In vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gC.

Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack.

Animals↗
PubMed1999

Novel mutations in the LKB1/STK11 gene in Dutch Peutz-Jeghers families.

The Peutz-Jeghers syndrome (PJS) is a rare hereditary disorder in which gastrointestinal hamartomatous polyposis, mucocutaneous pigmentation, and a predisposition for developing cancer are transmitted in an autosomal dominant fashion. The recently identified LKB1/STK11 gene located at chromosome 19p13.3 is mutated in a number of PJS pedigrees. We performed mutation analysis in 19, predominantly Dutch, PJS families. In 12 of these families, we identified LKB1/STK11 mutations, none of which has been described before. These 12 novel LKB1/STK11 mutations consist of one nonsense mutation, three frameshift deletions, three frameshift insertions, two acceptor splice site mutations, and three missense mutations. In addition, we detected four polymorphisms in LKB1/STK11. In the remaining seven PJS families, we found no apparent abnormalities of the LKB1/STK1I gene, which could reflect the existence of locus heterogeneity in PJS. None of the mutations occurred in more than one family, and a number were demonstrated to have arisen de novo. The diverse array of mutations found, the apparent high mutation rate, as well as the existence of a possible second PJS locus, renders diagnostic or predictive genetic testing in individual patients difficult, although future identification of additional mutations or even gene(s) will help in increasing the yield of direct mutation analysis.

AMP-Activated Protein Kinase Kinases↗
PubMed1999

Suspected hereditary nonpolyposis colorectal cancer: International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC) criteria and results of genetic diagnosis.

PURPOSE: The aim of this study was to determine the frequency of mutations in the mismatch repair genes in families suspected of having hereditary nonpolyposis colorectal cancer. METHODS: We devised two criteria for families suspected of having hereditary nonpolyposis colorectal cancer (Criteria I and II). Criteria I consist of at least two first-degree relatives affected with colorectal cancer with at least one of the following: development of multiple colorectal tumors including adenomatous polyp, at least one colorectal cancer case diagnosed before the age of 50, and occurrence of a hereditary nonpolyposis colorectal cancer extracolonic cancer (endometrium, urinary tract, small intestine, stomach, hepatobiliary system, or ovary) in family members. Criteria II consist of one colorectal cancer patient with at least one of the following: early age of onset (<40 years); endometrial, urinary tract, or small intestine cancer in the index patient or a sibling (one aged <50 years); and two siblings with other integral hereditary nonpolyposis colorectal cancer extracolonic cancers (one aged <50 years). A questionnaire was mailed to members of the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer to determine the mutation detection rate in mismatch repair genes from the families fulfilling these criteria. For comparison the mutation detection rate for families fulfilling the Amsterdam hereditary nonpolyposis colorectal cancer criteria in each institution was also obtained. RESULTS: Data were obtained from eight different institutions (in 7 different countries). In a total of 123 patients from 123 families (67 families fulfilling Criteria I and 56 families fulfilling Criteria II), genetic testing for germline mismatch repair gene variants was performed. Germline mutations of the hMLH1 or hMSH2 genes were identified in 24 families (20 percent). Of these, the mutation detection rate for families fulfilling Criteria I was 28 percent (19/67). The mutation detection rate for families fulfilling Criteria II was 9 percent (5/56). In these eight institutions, the overall mutation detection rate for families fulfilling the Amsterdam hereditary nonpolyposis colorectal cancer criteria was 50 percent (77/154). CONCLUSION: The Criteria I for suspected hereditary nonpolyposis colorectal cancer have the advantages that they can be applied to nuclear families and they can include extracolonic cancers. The results of this study suggest that families fulfilling Criteria I should be offered genetic testing. The relatively low mutation detection rate in those families fulfilling Criteria II suggests that, using current techniques, genetic testing in these families is not practical.

Age of Onset↗
PubMed1999

Loss or reduction of Fhit expression in renal neoplasias: correlation with histogenic class.

Involvement of the 3p14.2 region of chromosome 3 in kidney cancers was suggested 20 years ago, when a reciprocal constitutional translocation, t(3;8)(p14.2;q24), was shown to segregate with bilateral clear cell renal carcinoma in 3 generations of 1 family. The FHITgene that is interrupted at 3p14.2 by the t(3;8) translocation has been isolated, characterized, and shown to be frequently altered, mainly by internal deletion, in carcinomas or cancer-derived cell lines of the lung, stomach, pancreas, esophagus, cervix, and colon. Although up to 90% of sporadic clear cell renal carcinomas, representing 70% of adult renal carcinomas, exhibit loss of FHIT alleles, FHIT gene alterations have been documented for only a few renal cell carcinoma-derived cell lines. Nevertheless, more than 50% of clear cell carcinomas were recently shown to express little or no Fhit protein, unlike the normal kidney tubule epithelium, which is uniformly strongly positive for Fhit expression. We have extended our immunohistochemical study of expression of Fhit protein to the spectrum of histopathologic subtypes of adult renal tumors. There is an apparent continuum of Fhit expression from the 100% strongly positive oncocytomas through mostly positive papillary and chromophobe to the mostly negative clear cell and sarcomatoid to the negative or predominantly negative collecting duct renal carcinomas. This pattern of diminishing Fhit expression correlates with reported frequency of 3p allele loss in renal carcinomas and may parallel the potential for aggressive behavior of tumors, as suggested by the abundant Fhit expression in the benign oncocytomas and the near absence of Fhit expression in sarcomatoid and collecting duct RCCs.

Acid Anhydride Hydrolases↗
PubMed1999

Molecular evidence for derivation of metastatic cells from minor subclones of primary clear renal cell carcinomas.

The production of metastases depends on changes in a large number of genes. It is also connected with the interaction of tumor cells with the environment. It has been reported that primary tumor clone domination is also an important factor in metastasizing, and in many neoplasms dominating clones are the metastatic forerunners. Up to now it is unknown whether domination of a given clone in a primary renal cell carcinoma is a crucial factor in forming metastases or rather presence or absence of specific genes imposes the major advantage in the metastatic process. To study the presence or absence of the duplication and mitotic nondisjunction event as one of the phenomenon in the creation of metastases, as well as possible derivation of metastatic cells from the minor subclone of primary tumor, we examined three metastatic renal clear-cell carcinomas in which by comparative genomic hybridization we detected the loss of 3p in the primary tumor and two copies of 3p in the corresponding metastasis. Loss of heterozygosity analyses using markers for 3p25 (D3S1038), 3p21.1 (D3S1295), and 3p14.2 (D3S1481) proved heterozygosity of at least two 3p loci in all metastatic tumors, which indicates the absence of mitotic nondisjunction event as a cause of occurrence of two copies of 3p in metastases. Our results suggest that in some of the clear-cell renal carcinomas metastatic cells may derive from minor subclones of primary tumors.

Adrenal Gland Neoplasms↗
PubMed1998

Absence or reduction of Fhit expression in most clear cell renal carcinomas.

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.

Acid Anhydride Hydrolases↗
PubMed1998

Viral interference with antibody and complement.

Viruses have evolved strategies to evade immunity mediated by antibody and complement. Herpesviruses and coronaviruses encode IgG Fc binding proteins that inhibit IgG activity, enabling the virus or infected cell to escape antibody attack. Herpesviruses, vaccinia virus and HIV-1 have the capacity to interfere with complement, either by incorporation of cellular complement regulatory proteins into the virion envelope or cell membrane, or by expression of viral molecules that mimic functions of complement regulatory proteins. The structure and biological activities of herpes simplex virus type 1 (HSV-1) glycoproteins gE, gI and gC are described. These glycoproteins protect HSV from immune attack; HSV-1 gE/gI form a complex that binds the Fc domain of IgG while gC is a C3b binding complement regulatory protein, providing a survival advantage to the virus in vitro and in vivo by inhibiting immune functions.

Animals↗
PubMed1998

In vivo immune evasion mediated by the herpes simplex virus type 1 immunoglobulin G Fc receptor.

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcgammaR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcgammaR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcgammaRs. NS-gE339 expresses gE and gI, is FcgammaR-, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcgammaR does not bind murine IgG; therefore, the absence of an FcgammaR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcgammaR- mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcgammaR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcgammaR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.

Animals↗
PubMed1997

Comparison of DNA gains and losses in primary renal clear cell carcinomas and metastatic sites: importance of 1q and 3p copy number changes in metastatic events.

Archival material from primary and metastatic renal clear cell carcinomas of 25 patients was studied by comparative genomic hybridization. Copy number changes of entire chromosomes or chromosomal subregions were detected in 22 primary and 21 metastatic tumors. Copy number changes affected the following chromosomes in at least 20% of the 25 primary tumors (minimal common region given in parentheses): gains were noted for chromosomes 1 (1q21-->q23), 5 (5q31-->q34), 7 (7p), 8 (8q), 16 (16p), 17 (17q12-->qter), 19, and 22 (22q12-->qter); losses were revealed for chromosomes 3 (3p21-->pter), 8 (8p23-->pter), 14(14q21-->qter), and Y. The same chromosomal regions that were involved in primary renal clear cell carcinomas were also found in the respective metastatic tumors but with strikingly different frequencies for a few regions. Metastatic tumors showed a significantly higher frequency of complete or partial gains of the long arm of chromosome 1, in particular at 1q21-->q23 than primary tumors (16 cases versus 6 cases; P < 0.005). These data suggest a correlation of metastatic events in renal clear cell carcinomas with an increase in the copy number of genes located at 1q, in particular at 1q21-->q23. In contrast, the entire or partial loss of the short arm of chromosome 3 was significantly less frequent in metastatic tumors (8 cases versus 15 cases; P < 0.025). The validity of 1q and 3p copy number changes detected by comparative genomic hybridization was confirmed by interphase cytogenetics with region-specific yeast artificial chromosomes to paraffin-embedded tumor tissue sections.

Adenocarcinoma, Clear Cell↗
PubMed1996

Losses at 3p common deletion sites in subtypes of kidney tumours: histopathological correlations.

Deletions of the short arm of chromosome 3 (3p) have been recognized as characteristic features of clear cell renal cell carcinomas (clear cell RCC). We analysed 55 clear-cell RCCs and 30 non-clear-cell kidney tumours (10 papillary and 7 chromophobic RCCs, 11 oncocytomas and 2 collecting duct carcinomas) in loss of heterozygosity (LOH) studies using microsatellite markers for previously observed regions of common deletions on 3p in kidney tumours (3p25, 3p21.3, 3p14.2 and 3p12-13). Alterations were found in all 55 cases of clear-cell RCCs at two to four of the 3p regions. Extensive losses were not found in non-clear-cell tumours except for collecting duct carcinomas; 1 of 10 papillary RCCs showed interstitial deletion limited to a single 3p21.3 locus. LOH analyses using microsatellite markers for regions of common deletions at 3p may be of value in differential diagnosis of kidney tumours.

Adenocarcinoma, Clear Cell↗
PubMed1996

Immune evasion properties of herpes simplex virus type 1 glycoprotein gC.

Herpes simplex virus type I (HSV-1) glycoprotein gC binds complement component C3b, and purified gC inhibits complement activation. Two HSV strains carrying mutations in the gC gene which rendered them unable to bind C3b were compared with wild-type and marker-rescued viruses to evaluate the role of gC on the virion in protecting HSV-1 from complement-mediated neutralization. The gC mutant viruses were markedly susceptible to neutralization by nonimmune human serum, showing up to a 5,000-fold decline in titer after 1 h of incubation with serum. In contrast, wild-type or marker-rescued viruses showed a twofold reduction in titer. Studies with hypogammaglobulinemic and immunoglobulin G-depleted serum supported the observation that neutralization occurred in the absence of antibody. Neutralization of gC mutant strains by nonimmune serum was rapid; their half-life was 2 to 2.5 min, compared with 1 h for wild-type virus. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated human serum or C4-deficient guinea pig serum failed to neutralize gC mutant strains, indicating a role for components of the classical complement pathway. gC had little additional effect on neutralization by the combination of antibody plus complement compared with complement alone. The results indicate that the magnitude of the protection offered by gC-1 is larger than previously recognized; that in the absence of gC-1, complement neutralization is rapid and is mediated by components of the classical complement pathway; and that gC mainly protects against antibody-independent complement neutralization, suggesting a probable role for gC early in infection, before antibodies develop.

Animals↗
PubMed1996

Detection of specific genetic alterations in cancer cells.

The genetic changes found in human neoplasms suggest that hematopoietic tumors develop mainly from inappropriate expression (usually overexpression) of a growth promoting gene (oncogene). In contrast, the progression to malignancy of carcinomas occurs mainly through loss of tumor suppressor genes. Gene therapy might be used to turn off an activated oncogene, eg, by antisense treatment, whereas gene therapy to overcome tumor suppressor gene loss necessarily would focus on gene replacement in the tumor cell or pharmacologically substituting for lost gene function. On the other hand, the protein products of mutations that activate oncogenes or that inactivate tumor suppressor genes are both potential tumor antigens. Increasingly, characterization of the molecular changes that contribute to the malignant phenotype provides information impacting on tumor diagnosis and patient prognosis.

Base Sequence↗