Characterization of D6S2806 and D6S2879 short tandem repeat loci in HLA-DRB1 region in Iranian population.

Genomewide screen analysis has shown the close association of the human leukocyte antigen (HLA)-DRB1 region with susceptibility to multiple sclerosis and a number of autoimmune diseases. Using bioinformatics software, several potential short tandem repeat (STR) markers have been introduced in this region in the major histocompatibility complex data base (dbMHC). In this study, the identity and characteristics of two putative STR markers, D6S2879 and D6S2806, in this region were examined in Iranian population. The loci were genotyped in 85 individuals using polymerase chain reaction followed by polyacrylamide gel electrophoresis and sequencing. Analysis of the allelic frequency showed the presence of six and four alleles for D6S2806 and D6S2879, respectively. Analysis of deviations from Hardy-Weinberg equilibrium (HWE) showed that D6S2806 was in equilibrium (P > 0.05). However, the D6S2879 locus showed a significant deviation from HWE (P < 0.05). Therefore, the D6S2806 locus could be suggested as a marker for linkage analysis and disease-susceptibility investigations in the MHC-DRB1 gene region.

The promyelocytic leukemia protein interacts with Sp1 and inhibits its transactivation of the epidermal growth factor receptor promoter.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth- and transformation-suppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between -150 and -16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitro-translated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.

The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases.

The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARalpha encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein.

The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARalpha gene by t(15;17) translocation. There is significant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-finger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARalpha was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARalpha, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.

Identification of 3 beta-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: association of autoantibodies with endocrine autoimmune disease.

Autoantibodies directed against steroid hormone-producing cells (SCA) detectable by immunofluorescence are typically found in a small proportion of patients with premature ovarian failure (POF) as well as in other endocrine autoimmune diseases. The SCA pattern stains cells in the outer zones of the adrenal cortex, ovary, and testis. To identify the molecular target of SCA, an adrenal complementary DNA expression library was screened using SCA-positive serum, and the steroid enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was identified. Only 1 of 48 (2%) patients with idiopathic POF, not pre-selected for the presence of other autoimmune diseases, had SCA by immunofluorescence, whereas 10 of 48 (21%) had anti-3 beta HSD autoantibodies detectable by immunoblot using recombinant human enzyme compared with 6 of 115 (5%) control subjects (P = 0.002). Absorption of SCA-positive serum with recombinant human 3 beta HSD abolished the immunofluorescence pattern. We also examined the prevalence of anti-3 beta HSD autoantibodies in other endocrine autoimmune diseases. Two of 112 (2%) diabetic patients, but none of the thyroid or Addisonian patients, had SCA by immunofluorescence. Twenty-six (23%) diabetic subjects (P < 0.001 vs. controls), 3 of 18 thyroid patients (P > 0.05 vs. controls), and none of 4 Addisonian patients had anti-3 beta HSD autoantibodies. 3 beta HSD is the first steroid cell autoantigen defined at the molecular level to be associated with idiopathic POF occurring in the absence of other polyglandular diseases. Autoantibodies to 3 beta HSD in patients with other organ-specific autoimmune diseases indicate that the enzyme behaves as a typical target of polyendocrine autoimmunity. Anti-3 beta HSD autoantibodies in patients with POF may provide a marker of those subjects whose ovarian failure is autoimmune in origin and, as recent studies suggest, may be salvageable with glucocorticoid treatment.

Collagen type VI gene expression in the skin of trisomy 21 fetuses.

OBJECTIVE: To determine whether the mechanism for the retention of interstitial fluid in trisomy 21 fetuses presenting with nuchal translucency at 10-14 weeks' gestation is an alteration in the composition of collagen type VI, which is normally a triple helix formed of three single chains, alpha1, alpha2, and alpha3. The genes responsible for the alpha1 and alpha2 chains, COL6A1 and COL6A2, are located on chromosome 21 and therefore may be overexpressed in trisomy 21, whereas COL6A3 is located in chromosome 2. METHODS: Skin tissue was obtained after termination of pregnancy at 11-16 weeks' gestation in five fetuses with trisomy 21 and five normal controls. Total RNA was extracted and the steady-state levels of COL6A1 and COL6A3 mRNA expression of the gene transcripts were determined. Additionally, the distribution of collagen type VI in the skin of trisomy 21 and normal fetuses was analyzed using an immunohistochemical method. RESULTS: The ratio of the normalized densitometric scores for the mRNA expression of COL6A1 to COL6A3 in the skin of trisomy 21 fetuses was twice as high as in normal fetuses. Immunohistochemistry demonstrated that in trisomy 21 fetuses collagen type VI formed a dense network extending from the epidermal basement membrane to the subcutis, whereas in normal fetuses dense staining was confined to the upper region of the dermis. CONCLUSION: The distribution for collagen type VI is different from normal in the skin of trisomy 21 fetuses, and there is overexpression of COL6A1 compared with COL6A3.

Genetic Diversity and Balancing Selection within the Human Phenylalanine Hydroxylase (PAH) Gene Region in Iranian Population.

BACKGROUND: Genetic diversity of three polymorphic markers in the phenylalanine hydroxylase (PAH) gene region including PvuII (a), PAHSTR and MspI were investigated. METHODS: Unrelated individuals (n=139) from the Iranian populations were genotyped using primers specific to PAH gene markers including PvuII(a), MspI and PAHSTR. The amplified products for PvuII(a), MspI were digested using the appropriate restriction enzymes and separated on 1.5% agarose. The PAHSTR alleles were identified using polyacrylamide gel electrophoresis followed by silver staining. The exact size of the STR alleles was determined by sequencing. The allele frequency and population status of the alleles were estimated using PHASE, FBAT and GENEPOP software. RESULTS: The estimated degree of heterozygosity for PAHSTR, MspI and PvuII (a) was 66%, 56% and 58%, respectively. The haplotype estimation analysis of the markers resulted in nine informative haplotypes with frequencies ≥5%. Moreover, the results obtained from Ewens-Watterson test for neutrality suggested that the markers were under balancing selection in the Iranian population. CONCLUSION: These findings suggested the presence of genetic diversity at these three markers in the PAH gene region. Therefore, the markers could be considered as functional markers for linkage analysis of the PAH gene mutations in the Iranian families with the PKU disease.

Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells.

Our previous studies demonstrated that PML is a growth suppressor that suppresses oncogenic transformation of NIH/3T3 cells and rat embryo fibroblasts. PML is a nuclear matrix-associated phosphoprotein whose expression is regulated during the cell cycle. Disruption of PML function by t(15;17) in acute promyelocytic leukemia (APL) plays a critical role in leukemogenesis. To further study the role of PML in the control of cell growth, we have stably overexpressed PML protein in the HeLa cell line. This overexpression of PML significantly reduced the growth rate of HeLa cells and suppressed anchorage-independent growth in soft agar. We consequently investigated several parameters correlated with cell growth and cell cycle progression. We found that, in comparison with the parental HeLa cells, HeLa/PML stable clones showed proportionally more cells in G1 phase, fewer cells in S phase and about the same number in G2/M phase. The HeLa/PML clones showed a significantly longer doubling time as a result of a lengthening of the G1 phase. No effect on apoptosis was found in HeLa cells overexpressing PML. This observation indicates that PML suppresses cell growth by increasing cell cycle duration as a result of G1 elongation. To further understand the mechanism of the effect of PML on HeLa cells, expression of cell cycle-related proteins in HeLa/PML and parental HeLa cells was analyzed. We found that Rb phosphorylation was significantly reduced in PML stable clones. Expression of cyclin E, Cdk2 and p27 proteins was also significantly reduced. These studies indicate that PML affects cell cycle progression by mediating expression of several key proteins that normally control cell cycle progression. These results further extend our current understanding of PML function in human cells and its important role in cell cycle regulation.

Transcriptional repression by the promyelocytic leukemia protein, PML.

Acute promyelocytic leukemia is characterized by the presence of a t(15; 17) chromosomal translocation which results in the expression of a chimeric gene product, PMLRAR alpha, consisting of an N-terminal-truncated retinoic acid receptor-alpha fused to a C-terminal-truncated PML. Several structural features, and regions of homology to known transcription factors, suggest that PML may be involved in the regulation of gene expression. In this study we have analyzed the transcriptional regulatory activity of PML using chimeric GAL4/PML constructs and GAL4-responsive reporter plasmids. The data presented demonstrate that PML, when fused to the DNA-binding domain of GAL4 (GAL4/PML), inhibits transcription from GAL4-responsive promoters. The magnitude of this repression is cell type and promoter dependent, and deletion studies show that the putative coiled-coil and part of the serine-rich regions of PML are required for this activity. These regions are also shown to be responsible for the repression of transcription activity from the EGFR promoter. The data presented also demonstrate that GAL4/PML can recruit PMLRAR alpha resulting in the retinoid-inducible transcriptional activation of a GAL4-responsive promoter, a function dependent on the presence of the coiled-coil region of PMLRAR alpha.

Efficient retroviral infection of mammalian cells is blocked by inhibition of poly(ADP-ribose) polymerase activity.

Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood. However, the endonuclease and DNA strand-joining activities of the virus-encoded integrase protein (IN) are thought to act in concert with other, as-yet-unidentified, endogenous nuclear components which are involved in the DNA repair process. The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which is dependent on DNA strand breaks for its activity, is involved in the efficient repair of DNA strand breaks, and maintenance of genomic integrity, in nucleated eukaryotic cells. In the present work, we examine the possible involvement of PARP in the retroviral life cycle and demonstrate that inhibition of PARP activity, by any one of three independent mechanisms, blocks the infection of mammalian cells by recombinant retroviral vectors. This requirement for PARP activity appears to be restricted to processes involved in the integration of provirus into the host cell DNA. PARP inhibition does not affect viral entry into the host cell, reverse transcription of the viral RNA genome, postintegration synthesis of viral gene products, synthesis of the viral RNA genome, or the generation of infective virions. Therefore, efficient retroviral infection of mammalian cells is blocked by inhibition or PARP activity.