Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism.

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.

Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth.

Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.

CK2 abrogates the inhibitory effects of PRH/HHEX on prostate cancer cell migration and invasion and acts through PRH to control cell proliferation.

PRH/HHEX (proline-rich homeodomain protein/haematopoietically expressed homeobox protein) is a transcription factor that controls cell proliferation, cell differentiation and cell migration. Our previous work has shown that in haematopoietic cells, Protein Kinase CK2-dependent phosphorylation of PRH results in the inhibition of PRH DNA-binding activity, increased cleavage of PRH by the proteasome and the misregulation of PRH target genes. Here we show that PRH and hyper-phosphorylated PRH are present in normal prostate epithelial cells, and that hyper-phosphorylated PRH levels are elevated in benign prostatic hyperplasia, prostatic adenocarcinoma, and prostate cancer cell lines. A reduction in PRH protein levels increases the motility of normal prostate epithelial cells and conversely, PRH over-expression inhibits prostate cancer cell migration and blocks the ability of these cells to invade an extracellular matrix. We show that CK2 over-expression blocks the repression of prostate cancer cell migration and invasion by PRH. In addition, we show that PRH knockdown in normal immortalised prostate cells results in an increase in the population of cells capable of colony formation in Matrigel, as well as increased cell invasion and decreased E-cadherin expression. Inhibition of CK2 reduces PRH phosphorylation and reduces prostate cell proliferation but the effects of CK2 inhibition on cell proliferation are abrogated in PRH knockdown cells. These data suggest that the increased phosphorylation of PRH in prostate cancer cells increases both cell proliferation and tumour cell migration/invasion.

PRH/HHex inhibits the migration of breast and prostate epithelial cells through direct transcriptional regulation of Endoglin.

PRH/HHex (proline-rich homeodomain protein) is a transcription factor that controls cell proliferation and cell differentiation in a variety of tissues. Aberrant subcellular localisation of PRH is associated with breast cancer and thyroid cancer. Further, in blast crisis chronic myeloid leukaemia, and a subset of acute myeloid leukaemias, PRH is aberrantly localised and its activity is downregulated. Here we show that PRH is involved in the regulation of cell migration and cancer cell invasion. We show for the first time that PRH is expressed in prostate cells and that a decrease in PRH protein levels increases the migration of normal prostate epithelial cells. We show that a decrease in PRH protein levels also increases the migration of normal breast epithelial cells. Conversely, PRH overexpression inhibits cell migration and cell invasion by PC3 and DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. Previous work has shown that the transforming growth factor-ß co-receptor Endoglin inhibits the migration of prostate and breast cancer cells. Here we show that PRH can bind to the Endoglin promoter in immortalised prostate and breast cells. PRH overexpression in these cells results in increased Endoglin protein expression, whereas PRH knockdown results in decreased Endoglin protein expression. Moreover, we demonstrate that Endoglin overexpression abrogates the increased migration shown by PRH knockdown cells. Our data suggest that PRH controls the migration of multiple epithelial cell lineages in part at least through the direct transcriptional regulation of Endoglin. We discuss these results in terms of the functions of PRH in normal cells and the mislocalisation of PRH seen in multiple cancer cell types.

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients.

The T-cell oncogene Lim-only 2 (LMO2) critically influences both normal and malignant haematopoiesis. LMO2 is not normally expressed in T cells, yet ectopic expression is seen in the majority of T-acute lymphoblastic leukaemia (T-ALL) patients with specific translocations involving LMO2 in only a subset of these patients. Ectopic lmo2 expression in thymocytes of transgenic mice causes T-ALL, and retroviral vector integration into the LMO2 locus was implicated in the development of clonal T-cell disease in patients undergoing gene therapy. Using array-based chromatin immunoprecipitation, we now demonstrate that in contrast to B-acute lymphoblastic leukaemia, human T-ALL samples largely use promoter elements with little influence from distal enhancers. Active LMO2 promoter elements in T-ALL included a previously unrecognized third promoter, which we demonstrate to be active in cell lines, primary T-ALL patients and transgenic mice. The ETS factors ERG and FLI1 previously implicated in lmo2-dependent mouse models of T-ALL bind to the novel LMO2 promoter in human T-ALL samples, while in return LMO2 binds to blood stem/progenitor enhancers in the FLI1 and ERG gene loci. Moreover, LMO2, ERG and FLI1 all regulate the +1 enhancer of HHEX/PRH, which was recently implicated as a key mediator of early progenitor expansion in LMO2-driven T-ALL. Our data therefore suggest that a self-sustaining triad of LMO2/ERG/FLI1 stabilizes the expression of important mediators of the leukaemic phenotype such as HHEX/PRH.

Transcriptional repression in eukaryotes: repressors and repression mechanisms.

For many, if not most genes, the initiation of transcription is the principle point at which their expression is regulated. Transcription factors, some of which bind to specific DNA sequences, generally either activate or repress promoter activity and thereby control transcription initiation. Recent work has revealed in molecular detail some of the mechanisms used by transcription factors to bring about transcriptional repression. Some transcriptional repressor proteins counteract the activity of positively acting transcription factors. Other repressors inhibit the basal transcription machinery. In addition, the repression of transcription is often intimately associated with chromatin re-organisation. Many transcriptional repressor proteins interact either directly or indirectly with proteins that remodel chromatin or can themselves influence chromatin structure. This review discusses the mechanisms by which transcriptional repression is achieved and the role that chromatin re-organisation plays in this process.

Prevalence of anaemia and iron deficiency in three trimesters in Rural Vellore district, South India.

A multistage sampling technique was used to select 845 pregnant women from two blocks in Vellore district. Haematological measurement of haemoglobin (Hb) was done on all women and serum ferritin (SF) on a subsample of 445. The prevalence of anaemia (Hb <11 g/dl) was 56.6%, 70.2% and 69.5%, respectively among the first, second and third trimester women. The mean Hb of 10.7 g/dl was significantly higher among the first trimester than among the second and third trimester women, which was less than the recommended value of 11 g/dl. Iron deficiency (SF <12 microg/L) was significantly (P< 0.05) more among the third trimester women than among the first. The high prevalence of anaemia in each trimester in pregnancy indicates the need for iron supplementation as early as possible starting from the fourth month of pregnancy.

PRH represses transcription in hematopoietic cells by at least two independent mechanisms.

PRH (proline-rich homeodomain protein) is strongly expressed in the hematopoietic compartment. Here we show that PRH is a repressor of transcription in hematopoietic cells. A fragment of PRH that includes the homeodomain can bind to TATA box sequences in vitro and can also bind to the TATA box-binding protein. PRH represses transcription from TATA box-containing promoters in intact cells but does not repress transcription from a promoter lacking a TATA box. A mutation in the PRH homeodomain that blocks binding to DNA but that has little or no effect on binding to the TATA box-binding protein significantly reduces the ability of the protein to repress transcription and provides the first clear demonstration that a homeodomain can bring about transcriptional repression in vivo by binding to a TATA box. However, we also show that mutation of the PRH homeodomain does not block the ability of PRH to repress transcription when this protein is tethered upstream of the TATA box via a heterologous DNA-binding domain. PRH also contains an N-terminal proline-rich repression domain that is separate from the homeodomain. Deletion mapping suggests that this repression domain contains at least two regions that both independently contribute to transcriptional repression.

The homeodomain protein PRH influences the differentiation of haematopoietic cells.

Haematopoiesis involves the differentiation of a self-renewing stem cell into all of the lineages found in circulating blood. Myb-Ets transformed chicken blastoderm cells (MEPs) have many of the characteristics of multipotent haematopoietic cells and represent a useful model system for the study of haematopoiesis. The proline-rich homeodomain protein (PRH) has previously been shown to be expressed in the haematopoietic compartment. In this report we show that PRH mRNA and protein levels are down regulated as MEPs differentiate along the myelomonocytic and erythrocytic lineages. In contrast, PRH mRNA and protein levels remain high as MEPs differentiate toward the thrombocytic lineage. Over-expression of full length PRH in MEPs inhibits their transformation and/or proliferation. However, the over-expression of N-terminally truncated PRH proteins results in normally proliferating cells that are predominantly differentiated along the myelomonocytic and eosinophilic lineages. These results suggest that PRH plays a role in the proliferation and differentiation of haematopoietic cells.

Immunodiagnosis of human lymphatic filariasis: A review.

A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.

Changes in moisture content, mycoflora and aflatoxin content of rice bran during storage.

The changes in moisture content, storage mycoflora and aflatoxin B1 (AFB1) in bran from untreated or raw rice (Rr) and parboiled rice (Pbr) stored in small lots in polyethylene bags were studied at 15-day intervals up to 60 days, using five lots of each type of bran. Deterioration was more rapid with reference to all the three parameters, in Rr bran compared to Pbr bran, the former becoming completely overgrown and caked with fungi by the end of 60 days. Aspergillus flavus was the dominant fungus in Pbr bran, whereas A. candidus and Trichoderma viride were abundant in Rr bran. The frequency of incidence as well as concentration of AFB1 increased with storage time in both types of bran, but the rate of increase as well as overall concentration were much higher in Rr bran. Thus raw rice bran is unsuitable for prolonged storage.

The activation domain of a basic helix-loop-helix protein is masked by repressor interaction with domains distinct from that required for transcription regulation.

While there are many examples of protein-protein interactions modulating the DNA-binding activity of transcription factors, little is known of the molecular mechanisms underlying the regulation of the transcription activation function. Using a two-hybrid system we show here that transcription repression of the basic domain/helix-loop-helix factor PHO4 is mediated by complex formation with the PHO80 repressor. In contrast to other systems, such as inhibition of GAL4 by GAL80 or of p53 by MDM2, where repression is mediated by direct interaction at regions overlapping the transcription activation domain, interaction with PHO80 involves two regions of PHO4 distinct from those involved in transcription activation or DNA-binding and dimerization. The possibility that repression of PHO4 by PHO80 may represent a general mechanism of transcription control, including regulation of the cell-type-specific transcription activation domain of c-Jun, is discussed.

Transcription activation by Myc and Max: flanking sequences target activation to a subset of CACGTG motifs in vivo.

The Myc oncoprotein has been implicated in control of cell growth, division and differentiation. Although Myc contains a bHLH-LZ motif, it fails to bind DNA alone but can do so by forming heterodimers with an unrelated bHLH-LZ protein, Max. Max homodimers and Myc-Max heterodimers share the ability to bind CACGTG or CATGTG elements. Current models, based on experimentally induced overexpression of Myc and Max in mammalian cells, propose that Max-Max homodimers repress while Myc-Max heterodimers activate transcription through CACGTG binding sites. The interpretation of the results using mammalian cells is complicated by the presence of numerous unrelated CACGTG binding transcription activators and the existence of two alternative Max dimerization partners, Mad and Mxi-1. Thus, the mechanism whereby overexpression of Max leads to transcriptional repression remains to be established. Using a yeast system we show that Max homodimers have the potential to activate transcription through CACGTG motifs. Activation by Max requires DNA binding and amino acids outside the bHLH-LZ domain but is reduced compared with activation by Myc-Max heterodimers. Moreover, transcriptional activation by Myc-Max heterodimers, but not Max-Max homodimers, is strongly inhibited in vivo by specific sequences flanking the core CACGTG binding motif, presumably reflecting reduced DNA binding affinity. These results suggest a mechanism for directing the Myc-Max complex to a specific subset of CACGTG-containing target genes.

Gene-regulatory properties of Myc helix-loop-helix/leucine zipper mutants: Max-dependent DNA binding and transcriptional activation in yeast correlates with transforming capacity.

Max is a basic helix-loop-helix/leucine zipper (bHLH/LZ) protein that forms sequence-specific DNA-binding complexes with the c-Myc oncoprotein (Myc). Using Saccharomyces cerevisiae, we have shown that the Max bHLH/LZ domain enables Myc to activate transcription through CACGTG and CACATG sequences in vivo, and that the number and context of such sites determines the level of activation. In addition, we have used yeast to investigate the role of the Myc helix-loop-helix (HLH) and leucine zipper (LZ) motifs in mediating Max-dependent DNA-binding and transcriptional activation in vivo using HLH/LZ mutants generated by site-directed mutagenesis. The results show that, while both motifs are essential for Myc to activate transcription, helix 2 of the HLH together with the contiguous LZ suffice to mediate complex formation with Max, whilst helix 1 is essential for sequence-specific DNA binding of Myc-Max complexes. Furthermore, the ability of Myc HLH/LZ mutants to bind DNA and activate transcription in collaboration with Max correlates closely with their neoplastic transforming activity in higher eukaryotic cells.

C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif.

The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination. The b-HLH motif is also present in the S. cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5. In this report we show that the b-HLH region of PHO4 is sufficient to confer specific DNA-binding to the sequence CACGTG and, by comparison of the basic regions of PHO4 with those of other recently isolated CACGTG-binding proteins, we identify a specific subset of conserved amino acids in the basic region likely to confer DNA-binding specificity. On the basis of these observations we predict successfully the effect of substituting the PHO4 basic region with that from c-myc and show that the chimaeric protein activates transcription from the CACGTG elements present in the PHO5 UAS. From these data it is clear that the myc basic region confers specific binding to the sequence CACGTG.

Natural occurrence of toxigenic fungi and mycotoxins in rice bran.

Thirty four samples of rice bran, of which 9 were from raw (untreated) rice (RR) and 25 from parboiled rice (PbR) were collected from commercial rice mills in and around Madras and analysed for storage mycoflora and mycotoxins. Fungi of the Aspergillus flavus group were present in 29 of the 34 samples (8 from RR and 21 from PbR) in quantities ranging from less than 1-432 thousand propagules/g, though not always as the dominant mycoflora. Fungal numbers were usually higher in RR than in PbR samples. Five of the 9 RR samples and 6 of the 25 PbR samples were positive for aflatoxins. Among 29 isolates of A. flavus obtained from the bran samples, 16 isolates -6 from RR bran and 10 from PbR bran - were found to be toxigenic in vitro. Some isolates of A. candidus also seemed to produce aflatoxin and other fluorescent substances.

Mutational analysis of the nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli.

During anaerobic growth of E. coli, the FNR protein activates transcription initiation at the nirB promoter. After chemical synthesis using deliberately contaminated nucleotides, we isolated a series of recombinant plasmids with single point mutations or one base pair deletions in the nirB promoter. The effects of these alterations on the anaerobic induction of promoter activity were measured. Mutations that abolish anaerobic induction identify the -10 hexamer sequence whilst changes that allow reduced induction suggest positions involved in FNR binding. Comparison of the nucleotide sequence of the nirB promoter with other promoters that are regulated by FNR show clear homologies, suggesting consensus sequences for FNR binding sites, and confirming that some of the point mutations described here do indeed act by weakening FNR binding.

The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite.

Using recombinant DNA techniques, nested deletions have been made upstream of the Escherichia coli nirB transcription start site and their effects on the regulation of nirB promoter activity have been measured. Nucleotide sequences downstream of -73 are sufficient for FNR-dependent induction of activity by anaerobic growth conditions. However, nucleotide sequences between -87 and -149 are essential for further induction by nitrite in the growth medium. The nucleotide sequence at the galP1 CRP binding site located from -31 to -52 displays some similarities with the same region at the nirB promoter. When the galP1 sequence from -30 to -54 was replaced by the corresponding nirB sequence, expression from galP1 became inducible by FNR under anaerobic growth conditions.

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.