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D P Singal

Publications and source records attributed to D P Singal.

At least 19 recordsLinked to original sources
PubMed1998

Transfection of TAP 1 gene restores HLA class I expression in human small-cell lung carcinoma.

HLA class I antigens of the human major histocompatibility complex (MHC) play an important role in immune response. Consistent with their role in immune surveillance, these antigens are expressed on most cell types. However, a marked deficiency or lack of expression of these antigens has been observed in a variety of human neoplasms. We have shown that a number of class I-deficient human tumor cell lines, including small-cell lung carcinoma, lacked products of MHC-encoded TAP1 and LMP2 genes. Since a direct evidence for the role of these genes in class I expression in tumor cells is not available, in the present study we transfected class I-deficient human small-cell lung carcinoma cells with cDNAs corresponding to TAP1 gene and to LMP2 gene. Following transfection, tumor cells expressed products of the respective transfected gene. Cell-surface expression of class I molecules was, however, observed in cells transfected with TAP1, but not in tumor cells transfected with LMP2 gene. Our results provide conclusive evidence for a role of TAP1 gene in class I expression and suggest that transfection of TAP genes may be useful to upregulate class I expression in tumor cells. This strategy for restoration of class I expression by transfection of TAP genes is relevant for tumor rejection and/or abrogation of metastases formation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Peptide transport in human lymphoblastoid and tumor cells: effect of transporter associated with antigen presentation (TAP) polymorphism.

CD8+ T-cells recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. These peptides bind to MHC class I molecules in the endoplasmic reticulum (ER) lumen. Antigenic peptides are translocated from the cytosol to the lumen of ER by transporter associated with antigen presentation (TAP) proteins. In this study, it is shown that TAP1 polymorphism influences the peptide substrate specificity in human B-lymphoblastoid and tumor cell lines. TAP1A and 1C alleles specifically enhance translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1B allele does not show specificity for the peptide C-terminus. Human basophilic leukemia (Ku812), and hepatocellular carcinoma (PLC/PRF/5) cells express TAP1 molecules and exhibit TAP-mediated allele-specific peptide uptake after gamma-interferon (gamma-IFN) treatment. Ku812 cells express TAP1A and preferentially take up antigenic peptides with a basic C-terminus, however, PLC/PRF/5 cells with the TAP1B allele take up low but equivalent levels of peptides regardless of basic, acidic, or hydrophobic C-termini. Moreover, TAP2 polymorphisms have no influence on the peptide translocation in normal or tumor cell lines. In addition, Daudi, a beta2-microglobulin (beta2m) deficient human Burkitt lymphoma, cell line also showed TAP-dependent peptide uptake. Taken together, these results suggest that human TAP1 but not TAP2 polymorphisms influence the antigenic peptide transport and that this transport is independent of beta2m in this system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

DQCAR microsatellite polymorphism and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with HLA-DR genes that share a five amino acid sequence motif, QKRAA or QRRAA, from position 70 to 74 in the third hypervariable region of the DRbeta1 molecule. Since the associations between DRB1 genes and susceptibility to RA are incomplete, in this study we examined the CA repeat polymorphic marker DQCAR, located between DQA1 and DQB1 genes, alleles in 98 adult patients with seropositive RA and 100 normal healthy controls. The prevalence of the DQCAR 117 allele was significantly higher in RA patients as compared to normal controls. On the other hand, the frequency of DQCAR 99 was lower in patients than in normal subjects. Analysis of the data suggested that DRB1 genes sharing the QKRAA/QRRAA epitope have the primary association with disease susceptibility and DQCAR alleles do not provide an additional risk for the development of RA.

Adult↗
PubMed1998

D6S273 microsatellite polymorphism and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic articular inflammatory disease associated with HLA-DR genes that share a five amino acid sequence motif, QKRAA or QRRAA, from position 70 to 74 in the third hypervariable region of the DRB1 molecule. Since these associations between DRB1 genes and susceptibility to RA are incomplete, we examined the role of a CA repeat polymorphic microsatellite marker, D6S273, located between HSP70 and Bat2 genes in the class III region of MHC, in susceptibility to RA. Ninety-seven adult patients with seropositive RA and 100 normal healthy subjects were studied. Two D6S273 alleles (132 and 138) showed significant differences in their prevalence in RA patients as compared to normal controls; allele 132 was significantly higher in total patients and in DRB1 QKRAA/QRRAA epitope-positive patients, and allele 138 was significantly higher in QKRAA/QRRAA-negative patients. Analysis of data suggested that the association of D6S273 132 allele with RA was secondary to that of DRB1 genes. On the other hand, D6S273 138 allele showed primary association with RA susceptibility in QKRAA/QRRAA epitope-negative patients. The D6S273 138 allele thus provides an additional risk in RA susceptibility. The results in the present study therefore suggest that two regions in MHC, DRB1 and D6S273, contribute to susceptibility to RA.

Adult↗
PubMed1998

HLA-DM polymorphisms in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the contribution of HLA-DMA and DMB genes, which play a crucial role in the HLA class II restricted antigen presentation pathway, in susceptibility to rheumatoid arthritis (RA). METHODS: The distribution of DMA and DMB alleles was examined in patients with RA and in healthy subjects by oligotyping of PCR amplified genomic DNA with sequence specific oligonucleotide probes. RESULTS: There were no significant differences in the prevalence of DMA and DMB alleles in patients with RA as compared to healthy controls. In addition, no significant differences in frequencies of DMA and DMB alleles were observed in RA susceptibility epitope positive RA patients and controls. CONCLUSION: DMA and DMB genes do not appear to play a role in susceptibility to RA.

Alleles↗
PubMed1997

Halothane and isoflurane enhance melanoma tumour metastasis in mice.

PURPOSE: To investigate the incidence of tumour metastasis from B16 melanoma tumour cells in experimental animals following exposure to equipotent concentrations of halothane or isoflurane, and to differentiate if exposure to one anaesthetic resulted in greater metastases than the other. METHODS: Experimental animals (C57B1 mice), were randomized to receive 1.3 MAC hours of halothane or isoflurane anaesthesia. The control group of animals received oxygen alone under identical conditions. Fifteen minutes after completion of anaesthesia, control and experimental groups were given 1 x 10(5) B16 melanoma cells intravenously. After 21 days, all animals were autopsied, and the metastatic nodules in their lungs were counted. The difference in the numbers of metastatic nodules between control and experimental groups of animals was analyzed for significance by the Mann Whitney "U test". RESULTS: More metastases were observed in the animals exposed to halothane (37.28 +/- 5.08, P < 0.0001), or isoflurane anaesthesia (28.24 +/- 4.07, P < 0.0014) than in the control animals (12.22 +/- 1.52). CONCLUSION: Exposure to halothane or isoflurane anaesthesia increased the number of pulmonary metastases in C57B1 mice compared with the control groups but there was no difference in metastases among animals treated with halothane or isoflurane.

Anesthetics, Inhalation↗
PubMed1997

Transporter associated with antigen-processing (TAP) genes and susceptibility to tuberculoid leprosy and pulmonary tuberculosis.

We have studied TAP polymorphism in a panel of 40 healthy individuals, 57 patients with pulmonary tuberculosis (PTB) and 50 with tuberculoid (TT) leprosy from North India. Only TAP2-A/F occurred with a significantly increased frequency in PTB patients as compared to controls (82.5% vs. 52.5%, P < 0.002, Pc < 0.01) giving a high relative risk of 4.3. On the other hand, TAP2-B was significantly increased in TT leprosy as compared to controls (76% vs. 47.5%, Pc < 0.003, RR 3.5) particularly in patients positive for HLA-DR15 than controls carrying DR15 (77.5% vs. 50%, P < 0.03, RR = 3.4). Further, TAP2-B allele was positively associated with DR15 negative PTB patients as compared to the DR15 positive group (43.8% vs. 17.1%, P < 0.04, RR = 0.3). This study along with our earlier studies on HLA association in mycobacterial diseases suggests that in addition to HLA-DR15 alleles in the TAP2 region influence susceptibility to PTB and TT leprosy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Lack of HLA-DR2-associated DRB1 gene in a family.

HLA-DR51 haplotypes co-express one DRB1 (B1*15 or B1*16) gene and one DRB5 gene. These haplotypes also carry two pseudogenes (DRB6 and DRB9) During routine HLA typing for transplantation, we observed an unexpected DR51 haplotype in two subjects (mother and daughter) in a family. Serological typing showed that these subjects are positive for DR51 and DQ6 but negative for DR15 and DR16. Investigations of genomic DNA by molecular techniques showed that these individuals carry DRB5*0101, DRB6*02 and DQB1*0602 genes, whereas the DRB1 gene associated with either DR15 (B1*1501 to B1*1504) or DR16 (B1*1601 to B1*1606) was not detected in the mother and daughter. It is possible that the new haplotype, DQB1*0602, DRB6*02, DRB5*0101, arose by deletion of DR2-associated DRB1 gene.

Female↗
PubMed1996

Molecular basis for lack of expression of HLA class I antigens in human small-cell lung carcinoma cell lines.

HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and thus play an important role in immune surveillance of cells infected with virus or altered by malignant transformation. Immunochemical studies have demonstrated a marked deficiency or lack of expression of class I molecules on the surface of many different types of tumor cells. It is likely that this allows these cells to escape immune surveillance. In the present study, we examined the molecular basis for lack of expression of class I antigens in small-cell lung carcinoma cell lines. Our results demonstrate that these cell lines also lacked products of MHC-encoded proteasome subunit LMP2 and the putative peptide transporter TAP1. In contrast, LMP7 and TAP2 genes were expressed in these cell lines. Pulse-chase experiments showed that class I molecules were unstable and thus not transported to the cell surface from endoplasmic reticulum. Our results suggest that antigenic peptides were not available for binding to class I alpha chains due to lack of TAP1 and LMP2 gene products. Investigations of the regulatory mechanisms of TAP1 and LMP2 genes showed that the tumor cells lacked trans -regulatory nuclear protein(s), which binds to the interferon-gamma (IFN-gamma) response element (ISRE) in the TAP1, LMP2 bidirectional intergenic promoter. Treatment of tumor cells with IFN-gamma induced ISRE-binding nuclear protein(s) and resulted in expression of TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. Our data provide credence for a role of TAP and LMP genes in immune response.

Base Sequence↗
PubMed1996

Polymorphism in both X and Y box motifs controls level of expression of HLA-DRB1 genes.

The HLA class II antigens of the human major histocompatibility complex play an important role in immune response. The quality of the immune response is determined not only by polymorphisms in their coding region, but also by the level of their cell-surface expression which affects, for example, the extent of T-cell activation. We have previously described allelic polymorphisms in the upstream regulatory regions of HLA-DRB genes, which affected DNA-protein interactions and resulted in significantly different promoter strengths. In the present study, we investigated the effect of polymorphisms in the X and Y box motifs on the transcriptional activity of DRB1 gene promoters in the DR1, DR51, and DR53 haplotype groups. We used normal, chimeric, and mutated DRB promoters and compared their relative abilities to initiate transcription of the CAT reporter gene in human B-cell lines. The results show that polymorphisms in both the X1 and Y box motifs play a dominant role in the promoter strength. In the gel mobility shift assay, we observed differential ability of nuclear proteins that bind to the polymorphic X1 and Y box elements. The results in the present study confirm earlier data in that the nucleotide variation in the X1 box affects the level of expression of DRB1 genes. In addition, the present data demonstrate that polymorphism in the Y box, which affects the inverted CCAAT sequence, also plays a dominant role in the transcriptional activity of DRB1 promoters.

Base Sequence↗
PubMed1996

Markedly decreased expression of TAP1 and LMP2 genes in HLA class I-deficient human tumor cell lines.

HLA class I antigens of the human major histocompatibility complex play an important role in immune response. These molecules present foreign antigenic peptides to cytotoxic T lymphocytes and thereby play a role in the immune surveillance of cells infected with virus or other intracellular pathogens or altered by malignant transformation. A marked deficiency or lack of expression of these antigens has been reported in a variety of human neoplasms. In the present study, we examined the expression of class I alpha chain, beta 2-microglobulin, TAP (TAP1 and TAP2) and LMP (LMP2 and LMP7) genes in a number of human tumor cell lines including small-cell lung carcinoma, hepatocellular carcinoma, colon adenocarcinoma and basophilic leukaemia. These cell lines were deficient in expression of both class I alpha chain and beta 2-microglobulin gene products. In addition, these cell lines lacked the products of MHC-encoded proteasome subunit LMP2 as well as the putative peptide transporter TAP1 genes. In contrast, TAP2 and LMP7 genes were expressed in these cell lines. Treatment of cells with gamma-IFN markedly enhanced the expression of class I alpha chain, beta 2-microglobulin, TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. The upregulation of TAP1 and LMP2 expression is associated with increased class I expression, suggesting that endogenous antigens, e.g. tumor antigens, could be presented by class I molecules following treatment of tumor cells with gamma-IFN.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Major histocompatibility-encoded human proteasome LMP2. Genomic organization and a new form of mRNA.

LMP2 is one of the two proteasome subunits encoded by genes in the major histocompatibility complex class II region. Here we report the genomic organization of human LMP2 gene. Sequence analysis of polymerase chain reaction-amplified cDNA from a number of lymphoblastoid cell lines demonstrated two forms of LMP2 mRNA, one (LMP2.1) complete and homologous to the published LMP2 genomic sequence from cosmid clones, and the other (LMP2.s) a smaller transcript resulting from splicing of a 30-base pair fragment from the first exon. Antibodies to recombinant LMP2.s protein (22.3 kDa) were raised in rabbits. This anti-LMP2.s serum recognized both recombinant proteins (LMP2.1 = 23.3 kDa and LMP2.s = 22.3 kDa) and a single protein of 21.5 kDa molecular mass in lysates from human lymphoblastoid cell lines. Pulse-chase experiments demonstrated that LMP2 polypeptide also undergoes processing from 22.3- to 21.5-kDa protein when incorporated into proteasomes. These data suggest that the processing of human LMP2 subunit takes place both at the transcription and post-translational levels. Northern blot analysis showed that the LMP2 mRNA is expressed in lymphoblastoid cell lines and in fibroblasts following gamma-interferon induction, but not in brain, smooth muscle, fibroblasts (uninduced), and colon epithelial cells.

Amino Acid Sequence↗
PubMed1994

HLA-DQ and TAP2 genes in patients with insulin-dependent diabetes mellitus.

We examined the prevalence of HLA-DRB1, DQB1, DQA1 and TAP2 genes in children with insulin-dependent diabetes mellitus (type 1 diabetes). These HLA and TAP2 alleles were identified by dot-blot analysis of polymerase chain reaction (PCR)-amplified genomic DNA with sequence-specific oligonucleotide probes. The results show that those DQB1 alleles, which carry non-aspartic acid at position 57, in conjunction with DQA1 alleles carrying arginine at position 52, are strongly associated with susceptibility to type 1 diabetes. The prevalence of the TAP2* 0201 allele in diabetic patients was significantly lower than that in normal controls. Analysis of the data suggests that DQ alleles have the primary association with type 1 diabetes and that the association of TAP2 alleles with the disease is secondary.

ATP Binding Cassette Transporter, Subfamily B, Mem↗