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Biomedical subjects

P A Lazo

Publications and source records attributed to P A Lazo.

At least 19 recordsLinked to original sources
PubMed2005

Aberrant expression of tetraspanin molecules in B-cell chronic lymphoproliferative disorders and its correlation with normal B-cell maturation.

Tetraspanin proteins form signaling complexes between them and with other membrane proteins and modulate cell adhesion and migration properties. The surface expression of several tetraspanin antigens (CD9, CD37, CD53, CD63, and CD81), and their interacting proteins (CD19, CD21, and HLA-DR) were analyzed during normal B-cell maturation and compared to a group of 67 B-cell neoplasias. Three patterns of tetraspanin expression were identified in normal B cells. The first corresponded to bone marrow CD10(+) B-cell precursors (BCP) which showed high expression of CD81 and CD9, low reactivity for CD53 and negativity for CD37. CD10(-) B-lymphocytes showed downregulation of CD9/CD81 and upregulation of CD53/CD37. Plasma cells showed re-expressed CD9 and downregulated CD37. Hierarchical clustering analysis of flow cytometry immunophenotypic data showed a good correlation between the tumor differentiation stage and the pattern of tetraspanin expression, with all analyzed individual samples classified into three major groups, independently of their normal or neoplastic origin. Despite this, neoplastic B-cells frequently showed aberrantly high/low expression of the different markers analyzed. Interestingly, in B-cell chronic lymphocytic leukemia, abnormal expression of CD53 and CD9 were associated with different patterns of disease infiltration, which would support the role of these molecules on modulating adhesion and migration of neoplastic B cells.

Antigens, CD↗
PubMed2003

Human papillomavirus DNA in cervical lesions from Morocco and its implications for cancer control.

To determine the types of human papillomaviruses (HPV) in northern Morocco, information which is needed for the design and use of HPV vaccines, we have analysed 129 cervical biopsies from this region. In our study, 91 cases were HPV positive, 45 cases had HPV-16 DNA, and 20 cases had HPV-18 DNA. This distribution of virus type was similar in inflammatory cervical lesions and in invasive cervical carcinomas. In conclusion, the HPV type distribution in Morocco is similar to that in other African Mediterranean countries, where the proportion of HPV-18 cases is significantly higher than in Europe. Determination of virus-type distribution is essential for vaccination programs.

Biopsy↗
PubMed2001

Differential cooperation between regulatory sequences required for human CD53 gene expression.

CD53 is a tetraspanin protein mostly expressed in to the lymphoid-myeloid lineage. We have characterized the human CD53 gene regulatory region. Within the proximal 2 kilobases, and with opposite transcriptional orientation, is located the promoter-enhancer of a second gene, which does not affect CD53. Twenty-four copies of a CA dinucleotide repeat separate these two gene promoters. The proximal enhanceosome of the human CD53 gene is comprised between residues -266 and +84, and can be subdivided into four major subregions, two of them within exon 1. Mutational analysis identified several cooperating sequences. An Sp1 and an ets-1 site, at positions -115 and +62, respectively, are essential for transcriptional competence in all cell lines. Five other regulatory sequences have a dual role, activator or down-regulator, depending on the cell line. At the end of the non-coding exon 1, +64 to +83, there is a second ets-1 regulatory element, which is required for high level of transcription, in cooperation with the Sp1 site, in K562 and Molt-4, but not in Namalwa cells, where it functions as a repressor. This Sp1 site also cooperates with another ets-1/PU.1 site at -172. Different cell types use different regulatory sequences in the enhanceosome for the expression of the same gene.

Antigens, CD↗
PubMed2001

Genomic organization, chromosomal localization, alternative splicing, and isoforms of the human synaptosome-associated protein-23 gene implicated in vesicle-membrane fusion processes.

Synaptosome-associated protein-23 (SNAP23) is a component of the cellular mechanism required for specific membrane fusion and targetting of intracellular vesicles. We have cloned the full-length human cDNA and the SNAP23 gene. The SNAP23 gene has eight exons, with the initiation codon located in exon 2, and maps to the human chromosome 15q21-22 region. The human SNAP23 gene can generate two types of message, the full-length message (SNAP23A) and a shorter message (SNAP23B). The latter is the result of alternative splicing where exon 5 is joined to exon 7 and the skipping of exon 6; it thus lacks a region that is required for non-specific binding to plasma membranes. The two isoforms, expressed as fusion proteins with glutathione-S-transferase, interact in vitro with human syntaxin 6, thus retaining the specific protein interaction required for membrane fusion. Alterations in the SNAP23 gene might be involved in neurological and other diseases with defects in vesicle-membrane fusion processes that map to 15q15-21.

Alternative Splicing↗
PubMed2000

Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis.

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca(++) and GTP-gamma-S-induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti-syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE- t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.

Amino Acid Sequence↗
PubMed2000

The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein.

The tumour suppressor p53 protein integrates multiple signals regulating cell cycle progression and apoptosis. This regulation is mediated by several kinases that phosphorylate specific residues in the different functional domains of the p53 molecule. The human VRK1 protein is a new kinase related to a poxvirus kinase, and more distantly to the casein kinase 1 family. We have characterized the biochemical properties of human VRK1 from HeLa cells. VRK1 has a strong autophosphorylating activity in several Ser and Thr residues. VRK-1 phosphorylates acidic proteins, such as phosvitin and casein, and basic proteins such as histone 2b and myelin basic protein. Because some transcription factors are regulated by phosphorylation, we tested as substrates the N-transactivation domains of p53 and c-Jun fused to GST. Human c-Jun is not phosphorylated by VRK1. VRK1 phosphorylates murine p53 in threonine 18. This threonine is within the p53 hydrophobic loop (residues 13-23) required for the interaction of p53 with the cleft of its inhibitor mdm-2. The VRK1 C-terminus domain (residues 268-396) that contains a nuclear localization signal targets the protein to the nucleus, as determined by using fusion proteins with the green fluorescent protein. We conclude that VRK1 is an upstream regulator of p53 that belongs to a new signalling pathway.

3T3 Cells↗
PubMed2000

Amplification of human genomic sequences by human papillomaviruses universal consensus primers.

Detection of human papillomaviruses DNA (HPV) in tumour samples is often determined by a PCR based approach with standard universal consensus oligonucleotides. It is shown that these primers under the same amplification conditions can amplify a human genomic sequence of 1361 nucleotides in oral carcinomas and normal DNA samples. This sequence is detected more easily as the copy number of HPV DNA decreases. Therefore, in tumour samples that have a low copy number of HPV or that are contaminated by normal tissue there is a potential risk of misidentification of the presence of HPV if this observation is not taken into account.

Base Sequence↗
PubMed1999

Expression of a new isoform of the tumor susceptibility TSG101 protein lacking a leucine zipper domain in Burkitt lymphoma cell lines.

The tumor susceptibility gene, TSG101, has been identified as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283-1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with different dimerization potential opens up a new level of regulation of the TSG101 proteins possibly affecting cell cycle regulation.

Amino Acid Sequence↗
PubMed1999

Expression of aberrant functional and nonfunctional transcripts of the FHIT gene in Burkitt's lymphomas.

The 3p14.2 chromosome region, which contains the FHIT gene and the FRA3B fragile site, is frequently altered in carcinomas. We analyzed the expression of the FHIT gene in 21 Burkitt's lymphoma cell lines and normal lymphoid populations. Seventeen (80%) of these cell lines had a common aberrant FHIT transcript as well as the normal transcript. Exon 2 was often aberrantly spliced to several coding exons, skipping exons 3 and 4, which overlap FRA3B. Other aberrant transcripts lacked exons 4-7 or exons 5-8. Exon 5, which has the initiation codon, was the most commonly affected. In two cell lines, Raji and KK124, there were aberrant transcripts retaining only the coding exons, which were able to make a normal protein, as demonstrated by in vitro transcription-translation analysis. In these aberrant messages, the additional deletion of 11 nucleotides at the beginning of exon 10 resulted in loss of translation. The cell line Ramos did not have a normal transcript. Some transcripts had common insertions of unknown origin that replaced coding exons, mainly exons 6 and 7. None of these aberrant messages coded for a protein, whether normal or aberrant. Within an individual cell line, aberrant messages appeared to result from sequential splicing reactions of a transcriptional unit derived from one allele. There was no correlation between aberrant FHIT transcription and the type of Burkitt's lymphoma regarding chromosomal translocation or presence of Epstein-Barr virus. In normal tonsils, spleen, and peripheral blood lymphocytes, aberrant transcripts were not detected and might represent a very minor subpopulation if detectable.

Acid Anhydride Hydrolases↗
PubMed1999

Co-expression of several human syntaxin genes in neutrophils and differentiating HL-60 cells: variant isoforms and detection of syntaxin 1.

Syntaxins are major components of vesicle trafficking and their pattern of expression depends on the cell type. Using reverse transcriptase-polymerase chain reaction (RT-PCR), cloning, and sequencing techniques, we have found that human neutrophils and neutrophil-differentiated HL-60 cells co-express syntaxins 1A, 3, 4, 5, 6, 7, 9, 11, and 16. These genes are also expressed in human peripheral blood lymphocytes and SH-SY5Y neuroblastoma cells, which, unlike neutrophils, also expressed syntaxin 10. We have identified two isoforms of syntaxin 3. Syntaxin 3A, similar to the previously reported syntaxin 3, and the novel isoform syntaxin 3B, which is identical to syntaxin 3A but lacks 37 amino acid residues at the carboxy-terminal region. Syntaxin 1 was mainly located to neutrophil granule membranes by confocal microscopy and by immunoblotting of subcellular fractions. These data indicate that syntaxin 1 cannot be considered specific to neural tissues. The level of expression of syntaxins 3, 4, 6, and 11 was increased during neutrophil differentiation of HL-60 cells, whereas that of syntaxins 1A, 5, 9, and 16 was unchanged. Syntaxin 7 was not expressed in undifferentiated HL-60 cells, but its expression was induced on neutrophil differentiation. The expression of several syntaxin genes in human neutrophils could be related to the high secretory capacity of these cells as well as to the presence of different cytoplasmic granules with distinct exocytic capabilities.

Amino Acid Sequence↗
PubMed1999

The molecular genetics of cervical carcinoma.

In the pathogenesis of cervical carcinoma there are three major components, two of them related to the role of human papillomaviruses (HPV). First, the effect of viral E6 and E7 proteins. Second, the integration of viral DNA in chromosomal regions associated with well known tumour phenotypes. Some of these viral integrations occur recurrently at specific chromosomal locations, such as 8q24 and 12q15, both harbouring HPV18 and HPV16. And third, there are other recurrent genetic alterations not linked to HPV. Recurrent losses of heterozygosity (LOH) have been detected in chromosome regions 3p14-22, 4p16, 5p15, 6p21-22, 11q23, 17p13.3 without effect on p53, 18q12-22 and 19q13, all of them suggesting the alteration of putative tumour suppressor genes not yet identified. Recurrent amplification has been mapped to 3q+ arm, with the common region in 3q24-28 in 90% of invasive carcinomas. The mutator phenotype, microsatellite instability, plays a minor role and is detected in only 7% of cervical carcinomas. The development of cervical carcinoma requires the sequential occurrence and selection of several genetic alterations. The identification of the specific genes involved, and their correlation with specific tumour properties and stages could improve the understanding and perhaps the management of cervical carcinoma.

Chromosome Mapping↗
PubMed1998

Recurrent integration of papillomavirus DNA within the human 12q14-15 uterine breakpoint region in genital carcinomas.

Genital carcinomas are associated with human papillomaviruses, and the viral DNA is frequently integrated in the host cell genome. Recurrent chromosomal alterations are genetic markers for specific tumor phenotypes. To demonstrate that papillomavirus DNA integration is indeed a recurrent chromosomal aberration, we mapped two independent papillomavirus integration sites in the human 12q14-15 region, one containing HPV16 DNA and the other HPV18 DNA. The two HPV integration sites map approximately 10 kbp from each other within the cosmid LLNL12NCO1-196E1 clone. The integration site corresponding to HPV16 DNA in SK-v cells is proximal to the 5' end of a DNA segment known to be rearranged by integration of HPV18 DNA in another cervical carcinoma cell line, SW756. Both integrations are located in the PAL2 locus within the uterine leiomyoma cluster region of translocation.

Base Sequence↗
PubMed1998

Physiological activation of human neutrophils down-regulates CD53 cell surface antigen.

Tetraspanin transmembrane proteins have a metastasis suppressor effect by acting as cell motility brakes in tumor cells. CD53 is a panleukocyte antigen that belongs to the tetra-span superfamily. Human neutrophils express high levels of CD53. We tested the hypothesis that this antigen level changes when cells are activated. Treatment of human neutrophils with their physiological activators, tumor necrosis factor alpha or platelet-activating factor, resulted in down-regulation of this antigen from the cell surface, as assessed by immunofluorescence flow cytometry. Similar responses were observed when neutrophils were stimulated with chemotactic N-formyl-methionyl-leucyl-phenylalanine, phorbol ester, or the calcium ionophore ionomycin. The CD53 antigen down-regulation upon neutrophil stimulation was further confirmed by immunoblotting analysis and was not correlated with a change in the level of CD53 transcripts. This CD53 antigen down-regulation paralleled that of CD43 and CD44 antigens in these cells, despite their different protein structure. The down-regulation of the three antigens CD53, CD43, and CD44 could be inhibited by phenylmethylsulfonyl fluoride, suggesting that CD53 antigen down-regulation is the result of the activation of a proteolytic mechanism. Down-regulation of CD53 antigen level, as a result of cellular stimulation, might play a role in the different aspects of neutrophil biology, by modulating its interactions on the cell surface.

Antigens, CD↗
PubMed1998

Pattern of expression of tetraspanin antigen genes in Burkitt lymphoma cell lines.

Tetraspanin antigens are implicated in the prognosis of different types of tumours. In this study we determine by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) the level of 13 tetraspan messages in 21 Burkitt lymphoma (BL) cell lines. All tumour cell lines have a common pattern of tetraspanin gene expression. There are five antigens which are detected in 90% of cell lines at high levels, CD53, CD81, CD63, SAS and CD82. Another two, CD9 and CD37, were detected in 60% of cell lines, and have a very variable level of expression. The remaining antigens, A15, CoO29, KRAG, L6, TI-1 and il-TMP, are expressed at low levels in very few cell lines without any specific pattern. The level of gene expression corresponds with the level of cell surface antigen determined by flow cytometry. The average number of tetraspan proteins expressed per cell line is six. These proteins may form subunits of an oligomeric structure with 24 transmembrane domains. There are no major differences in tetraspan expression pattern among sporadic or endemic tumours, type of translocation or Epstein-Barr virus status, suggesting the original cell of these tumours is the same, probably a late pre-B cell, at the CD9 to CD37 transition point. Tetraspanin gene expression is consistent with BL being a single entity, despite variations in other parameters.

Antigens, CD↗
PubMed1997

Ligation of CD53/OX44, a tetraspan antigen, induces homotypic adhesion mediated by specific cell-cell interactions.

The CD53 antigen is a member of the tetraspan family of proteins with unknown function. Stimulation of rat IR938F B-cell lymphoma cells with monoclonal antibody MRC OX44 (anti-rat CD53) triggered a homotypic adhesion reaction which reached a maximum effect at 24 hr. This effect occurred at 37 degrees C but not at 4 degrees C. Adhesion was prevented by removal of divalent cations, Ca2+ and Mg2+, with EGTA and EDTA as chelating agents. The adhesion induced by MRC OX44 was inhibited by cycloheximide and actinomycin D, suggesting that de novo protein synthesis was required for this effect. The addition of mAb WT1 against rat LFA-1 (CD11a) antigen had no effect on adhesion, suggesting that the cell-cell interaction is not mediated by the expression of LFA-1 antigen. The intracellular signals required to induce adhesion were inhibited by two tyrosine kinase inhibitors, genistein and piceatannol. Wortmannin, a selective inhibitor of phosphoinositide 3-kinase activity, completely blocked adhesion. Two protein kinase C inhibitors, H7 and bisindolylmaleimide, inhibited the adhesion, suggesting that part of the signal is mediated by PKC. Electron microscopy of aggregated cells showed that the interaction is localized to short membrane regions, where contact areas of higher density in opposing zones from both cells were detected. We postulate that there is a common adhesion mechanism that is modulated by several tetraspan family members and associated proteins. This adhesion structure might represent a novel form of cell communication among lymphoid cells.

Animals↗