Terry Roberts

Connective Tissue Growth Factor and Regulation of the Mesangial Cell Cycle: Role in Cellular Hypertrophy

Connective tissue growth factor (CTGF) is now considered to be one of the important driver molecules for the pathogenesis of diabetic nephropathy (DN) and possibly many other fibrotic disorders. However, the molecular mechanisms by which CTGF functions remain to be established. In an attempt to define these mechanisms, this study was designed to investigate whether CTGF has any effect on the cell cycle of human mesangial cells (HMC), which are known to undergo hypertrophy in DN. This report provides the first evidence that CTGF is a hypertrophic factor for HMC. CTGF stimulates HMC to actively enter the G(1) phase from G(0), but they do not then progress further through the cell cycle. The molecular mechanisms underlying this G(1) phase arrest appear to be due to the induction of the cyclin-dependent kinase inhibitors (CDKI) p15(INK4), p21(Cip1), and p27(Kip1), which are known to bind and inactivate cyclinD/CDK4/6 and the cyclin E/CDK2 kinase complexes. This could account for the maintenance of pRb protein in a non- or very low-phosphorylated state, preventing cell cycle progression. Using CTGF antisense oligonucleotides, the results also indicate that the previously identified transforming growth factor-beta (TGF-beta)-induced hypertrophy in mesangial cells is CTGF-dependent. Mesangial cell hypertrophy is one of the earliest abnormalities of diabetic nephropathy; therefore, therapeutic strategies targeting CTGF may be beneficial in controlling DN.

The mRNA expression of SETD2 in human breast cancer: correlation with clinico-pathological parameters

BackgroundSET domain containing protein 2 (SETD2) is a histone methyltransferase that is involved in transcriptional elongation.There is evidence that SETD2 interacts with p53 and selectively regulates its downstream genes. Therefore, it could be implicated in the process of carcinogenesis. Furthermore, this gene is located on the short arm of chromosome 3p and we previously demonstrated that the 3p21.31 region of chromosome 3 was associated with permanent growth arrest of breast cancer cells. This region includes closely related genes namely: MYL3, CCDC12, KIF9, KLHL18 and SETD2. Based on the biological function of these genes, SETD2 is the most likely gene to play a tumour suppressor role and explain our previous findings.Our objective was to determine, using quantitative PCR, whether the mRNA expression levels of SETD2 were consistent with a tumour suppressive function in breast cancer. This is the first study in the literature to examine the direct relationship between SETD2 and breast cancer.MethodsA total of 153 samples were analysed.The levels of transcription of SETD2 were determined using quantitative PCR and normalized against (CK19).Transcript levels within breast cancer specimens were compared to normal background tissues and analyzed against conventional pathological parameters and clinical outcome over a 10 year follow-up period.ResultsThe levels of SETD2 mRNA were significantly lower in malignant samples (p = 0.0345) and decreased with increasing tumour stage.SETD2 expression levels were significantly lower in samples from patients who developed metastasis, local recurrence, or died of breast cancer when compared to those who were disease free for > 10 years (p = 0.041).ConclusionThis study demonstrates a compelling trend for SETD2 transcription levels to be lower in cancerous tissues and in patients who developed progressive disease. These findings are consistent with a possible tumour suppressor function of this gene in breast cancer.

Molecular characterization of prostate carcinoma tumor antigen-1, PCTA-1, a human Galectin-8 related gene

The galectin family of proteins has been associated with several diverse cellular processes. More than 30 years since the discovery of the first member, precise biological functions for the family as a whole, or for individual members has proven elusive. The isolation of Prostate Carcinoma Tumor Antigen-1 (PCTA-1), a cDNA closely related to rat and human Galectin-8, as a surface marker associated with prostate cancer was achieved using a previously described immunological subtraction approach, Surface Epitope Masking (SEM) approach, in combination with expression screening. It appears that PCTA-1 expression is almost ubiquitous in normal human tissues and could alter in specific contexts such as transformation or metastasis. Multiple expression isoforms of PCTA-1 at the mRNA level are observed. PCTA-1 maps to 1q42-43, a locus associated with predisposition to prostate cancer. We have determined the genomic structure of PCTA-1 to account for the several observed isoforms, performed expression analysis to determine distribution in normal and transformed contexts at the RNA and protein level and conducted over-expression studies to determine effects on cellular phenotype.

Molecular characterization of the 1p22 breakpoint region spanning the constitutional translocation breakpoint in a neuroblastoma patient with a t(1;10)(p22;q21).

To characterize the breakpoint in a neuroblastoma patient with a constitutional rearrangement we have constructed a yeast artificial chromosome (YAC) contig extending approximately 6 Mbp in the chromosome 1p22 region that spans the D1S435 and D1S236 loci. This contig has been confirmed by the coincidence of a number of markers in different overlapping YACs. For several of these YACs the overlap was demonstrated following the isolation and sequencing of end clones from which STS markers were generated. The majority of the YACs have been shown not to be chimeric either through the analysis of somatic cell hybrids or fluorescence in situ hybridization. Following the establishment of the contig we have been able to construct a physical map of the region that incorporates six STS and three newly assigned eSTS markers. The generation of this physical map has allowed the reordering of markers in the genetic linkage map for 1p. The physical order is; tel-D1S435-D1S188-D1S424-D1S236-D1D415- D1S420. With the reordering of D1S435 we have been able to join this contig with another reported previously, thereby generating a well characterized 15 Mbp YAC contig in the 1p22-31 region. The 6 Mbp contig described here spans the chromosome 1 constitutional translocation break-point seen in a patient with a t(1;10)(p22;q21) and who had a stage 4S neuroblastoma. YAC fragmentation has been used to define a 200 Kb region within this contig containing the 1p22 breakpoint. Restriction enzyme analysis demonstrates that there are three NotI sites in this region, one of which lies close to the translocation breakpoint site.

Fine structure physical mapping of a 1·9 Mb region of chromosome 13q12

Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13. Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies. In particular, seven cases of a t(8;13) (p11; q12·1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T‐cell leukemia and eosinophilia. We have previously identified a CEPH megaYAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation. As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1·9 Mb YAC. We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC. Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen‐NIB1257‐(ATP1AL1/D13S283)‐ D13S179E‐(D13S504E/D13S505E)‐D13S824E‐D13S182E‐D13S221‐tel. These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.

Cloning of the human Gfi-1 gene and its mapping to chromosome region 1p22

Recently the rat and mouse Growth Factor Independence (Gfi-1) genes have been cloned (Gilks et al., 1993; Zoring et al; 1996). This gene allows cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 which are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, we have identified the human homologue of the Gfi gene and defined a 50 Kb map position within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved.

Identification of subpopulations of cells with differing telomere lengths in mouse and human cell lines by flow FISH

Telomeres are specialized nucleoprotein structures at chromosome ends that undergo dynamic changes after each cell cycle. Understanding the mechanisms of telomere dynamics is critically dependent on the ability to accurately measure telomere length in a cell population of interest. Techniques such as Southern blot, which measures average telomere length, and quantitative fluorescence in situ hybridization (Q‐FISH), which can estimate telomere length in individual chromosomes, are limited in their capacity to determine the distribution of cells with differing telomere lengths in a given cell population.

The effect of chemotherapeutic agents on telomere length maintenance in breast cancer cell lines

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability.

Characterisation of a human chromosome 1 somatic cell hybrid mapping panel and regional assignment of 6 novel STS.

A somatic cell hybrid mapping panel has been constructed which allows subdivision of human chromosome 1 into 8 distinct subregions. All of the hybrids carry copies of chromosome 1 with specific deletions and the position of the breakpoints has been determined relative to the location of microsatellite markers in the genetic linkage map produced by Genethon. The majority of the breakpoints can be positioned between adjacent loci on the map. The usefulness of this hybrid panel for physical mapping has been demonstrated by the regional assignment of 6 novel STS markers made from Alu‐PCR clones generated from a hybrid which contains the short arm of chromosome 1.

An Activity Associated with Human Chromosome 21 Permits Nuclear Colocalization of the Adenovirus E1B-55K and E4orf6 Proteins and Promotes Viral Late Gene Expression

ABSTRACT The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.