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Biomedical subjects

S A Bustin

Publications and source records attributed to S A Bustin.

36 records · Page 2Linked to original sources
PubMed1999

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients.

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5'-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.

Adult↗
PubMed1999

The product of the primary response gene BRF1 inhibits the interaction between 14-3-3 proteins and cRaf-1 in the yeast trihybrid system.

The 14-3-3 proteins are small abundant cytosolic eukaryotic proteins that associate with and modulate the activity of numerous other proteins. The 14-3-3 beta isoform has been shown to bind to the product of the protooncogene cRaf-1 and to facilitate its activation by Ras. Using the yeast two-hybrid system, we have demonstrated that 14-3-3 beta and another isoform, 14-3-3 tau, bind to the product of the primary response gene BRF1 and that the interaction between each isoform and BRF1 is significantly stronger than that with cRaf-1. We further demonstrated that the charge of residue 187 in 14-3-3 beta regulates its affinity for both BRF1 and cRaf-1. The interaction of either isoform with BRF1 requires both proteins to be fully intact. When all three proteins are coexpressed in a yeast trihybrid system, BRF1 interferes significantly with the binding of 14-3-3 to full-length cRaf-1 as well as to its regulatory and kinase domains. Using quantitative reverse transcription-polymerase chain reaction, 14-3-3 beta and BRF1 were found to be coexpressed in four different human tissues, suggesting a biologic role for their interaction in the regulation of cRaf-1-mediated signal transduction processes.

14-3-3 Proteins↗
PubMed1998

An enzyme-linked immunosorbent assay for the detection of agents which interfere with the DNA binding activities of transcription factors--exemplified by NF-IL6.

Binding of transcription factors to DNA is usually detected by the electrophoretic mobility shift assay also known as gel-shift or band-shift assay. Recently the use of biosensors has allowed factor/DNA interactions to be followed in real time. However, neither of these approaches lends itself easily to high-throughput screening of agents which might interfere with this process. We have therefore developed a 96-well plate-based enzyme-linked immunosorbent assay for this purpose. Biotinylated oligonucleotides bound to streptavidin-coated plates are used to capture recombinant transcription factor proteins which can then be detected using specific antibodies which in turn are recognised by peroxidase-conjugated antisera. The peroxidase catalyzes conversion of a colorless substrate to a colored product which can be quantified by optical densitometry on a plate reader. Agents which interfere with the binding of the transcription factor to DNA reduce the optical density in the well. Using the NF-IL6 transcription factor we show that the assay can detect reductions to 86% at significant levels. The assay may be readily adapted for robotic manipulation, making it ideal for high-throughput screening.

Base Sequence↗
PubMed1998

Molecular assessment of tumour stage and disease recurrence using PCR-based assays.

Solid cancers arise as a consequence of the accumulation of genetic and epigenetic alterations within a single cell or group of cells. Their ongoing characterization is providing a range of acid-based molecular markers for neoplasia. This, together with continuous refinements to the polymerase chain reaction (PCR), had led to the emergence of PCR-based assays as potential aids in the clinical management of cancer patients. Although the sensitivity of molecular diagnosis has the potential to aid clinicians in therapeutic decision making, problems with its specificity mean that the predictive value of molecular staging is still unproved. Its role in the identification of minimal residual disease after curative surgical resection requires clinical validation in further prospective studies.

Humans↗
PubMed1998

Genetic detection of lymph node micrometastases in patients with colorectal cancer.

BACKGROUND: Undetected micrometastases represent the single most important cause of treatment failure in patients undergoing putatively curative resection for colorectal cancer because current staging techniques are unable to identify patients with minimal residual disease. METHODS: A highly sensitive reverse transcription-polymerase chain reaction technique has been used to amplify tissue-specific messenger RNA from lymph nodes classified as tumour-free using both conventional histopathology and immunohistochemistry. RESULTS: Four of 15 patients were restaged after genetic diagnosis of lymph node micrometastases, while in a further two additional positive nodes were detected. CONCLUSION: Sensitive genetic techniques that detect minimal residual disease merit further study, particularly as there is evidence that patients may benefit from adjuvant chemotherapy.

Adult↗
PubMed1998

Differential effects of sodium butyrate on the transcription of the human TIS11 family of early-response genes in colorectal cancer cells.

The tetradecanoyl phorbol acetate (TPA)-inducible sequence 11 (TIS11) family of early-response proteins consists of at least five members. They share a highly conserved Cys3His zinc-binding motif, but otherwise have little sequence similarity. Their function remains unknown, but all are induced rapidly and transiently in response to extracellular hormone and growth factor signals. Sodium butyrate, a fermentation product of dietary fibre, effects colorectal cancer cell proliferation by inducing growth arrest, differentiation and apoptosis. In this communication, we report that butyrate has differential effects on the transcription of the three human TIS11 family members identified so far in T84 and HT-29 human colorectal cancer cell lines. Butyrate response factor 1 (BRF1) transcription is repressed, butyrate response factor 2 (BRF2) transcription is activated and there is no apparent effect on the transcription of human TIS11 (HTIS11). Induction and repression occur rapidly, with altered mRNA levels detectable within 15 min of butyrate addition. Two other short-chain fatty acids, propionate and acetate, have no detectable effects on BRF1 or BRF2 transcription.

Butyrate Response Factor 1↗
PubMed1995

The human immediate early gene BRF1 maps to chromosome 14q22-q24.

BRF1 (Butyrate response factor 1) is a member of an immediate early gene family specifying putative nuclear transcription factors. A repeat motif incorporating two Cys and two His is highly conserved between family members identified from yeast, Drosophila, mouse, rat, and human. The chromosome localization of none of the human genes has been determined thus far. Using the polymerase chain reaction on a human-rodent hybrid panel, we have localized BRF1 to chromosome 14. This was confirmed by direct sequencing of the PCR fragment. Using fluorescence in situ hybridization, the chromosome localization of BRF1 was further determined as 14q22-q24.

Animals↗
PubMed1995

ERF-2, the human homologue of the murine Tis11d early response gene.

A human cDNA specifying a member of the Tis11 early response gene family was cloned and sequenced. The human gene differs from its mouse homologue by encoding an additional 97 amino acids at its C-terminal end. The sequence has transactivation-like motifs, an unusual Cys-Ser-Ala-rich motif and displays sequence similarity at the extreme C-terminal end with another Tis11 family member, ERF-1.

Amino Acid Sequence↗
PubMed1995

Transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) is a close relative of epidermal growth factor (EGF), the first polypeptide mitogen discovered in 1962 (Cohen, 1962). TGF alpha, like EGF, exerts its effect on cells through binding to the EGF-Receptor (EGF-R). Here we review the molecular and cell biology of TGF alpha before proceeding to describe our own work on signaling molecules induced in response to activation of the EGF-R.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of ERF-1, a human member of the Tis11 family of early-response genes.

Members of the Tis11 family of early-response genes are characterized by a high degree of sequence similarity around a putative zinc finger motif. They are induced by a variety of cell agonists and polypeptide mitogens, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). We describe the cloning and sequencing of a human member of this gene family, EGF-response factor 1 (ERF-1), the homolog of the mouse Tis11b/rat cMG1 genes. The human and rodent genes are similar, with 5' UTR, coding sequence, and 3' UTR highly conserved. The promoter/enhancer region and intron sequences contain multiple putative transcription factor binding motifs characteristic of early-response genes. Amino acid sequence comparison of the seven members of the Tis11 family cloned so far identifies a repeated consensus motif of (x+)YKTELC(x+)x5GxCxYGx(x+)CxFxH involving the potential zinc finger. Toward the carboxyterminal end is a region with a high percentage of prolines (15/73) and, partially overlapping, a serine-rich domain (20/54). These may be important as trans-activation and phosphorylation sites. The 3' untranslated region is unusually long, extending over 1,860 bp. The sequence immediately downstream from the translational stop codon has extensive secondary structure potential. The 3' UTR is 60% AT rich, but contains two GC rich (> 70%) regions. In addition there are multiple reiterations of a destabilization sequence, as well as a single UUAUUUAU motif characteristic of mRNAs specifying proteins involved in the inflammatory response. The mRNA contains a consensus polyadenylation signal.

Amino Acid Sequence↗
PubMed1994

Transcription factors: targets for new designer drugs.

Many diseases are the result of aberrant regulation of cell and tissue-specific gene expression. At the molecular level they are often characterised by disruption of the cell cycle through inappropriate activation or inactivation of key regulatory proteins, termed nuclear transcription factors (NFs). NF activation is modulated by several potent classes of drug, including steroids, retinoids, and immunosuppressants like cyclosporin A and FK506. While such drugs have wide application, they lack specificity and produce undesired side-effects. Recently, however, the three-dimensional structures of some NFs, as well as their molecular interactions with DNA targets, have been reported and several biochemical pathways involved in altered NF function identified. This is setting the stage for a rational approach to the design of drugs that act in a tissue- or disease-specific manner, target particular genes and proteins, and will combine increased efficacy with reduced side-effects.

Drug Design↗
PubMed1993

Synthesis of preparative amounts of biologically active interleukin-6 using a continuous-flow cell-free translation system.

Cell-free synthesis of preparative amounts of interleukin-6 (IL-6) in a wheat germ extract is described. Following transcription of uncapped mRNA by SP6 polymerase, protein was synthesized in a continuous flow of translation components. The system remained active for at least 50 h, producing 1.25 x 10(6) units of IL-6. Recovered IL-6 was > 80% pure, reacted with anti-IL-6 polyclonal antiserum in Western blots, and had a specific activity comparable to recombinant IL-6 purified from Escherichia coli. This is the first time biologically active IL-6 has been prepared in preparative amounts using a continuous-flow cell-free translation system and confirms the feasibility of using enhanced in vitro translation for rapid synthesis of proteins from cloned templates.

Animals↗
PubMed1993

The role of apoptosis (programmed cell death) in haemopoiesis and the immune system.

Apoptosis, or programmed cell death, is a series of controlled sequential events resulting in the demise of cells without invoking an inflammatory response. It is a naturally occurring process which maintains a cellular balance during both animal development and in the mature adult. Although first described 20 years ago, there is now renewed interest in this phenomenon, particularly in the light of our greater understanding of cellular signalling pathways and their genetic control. This is especially pertinent to haemopoiesis and the overall maintenance of a functional immune system. This review broadly covers the biochemical events of apoptosis and the recognition of apoptotic cells by phagocytes. Reference is made to the selective development of T- and B-cells and to the control of inflammation. Molecular events in apoptosis are also discussed with special reference to aberrant bcl-2 gene expression in follicular B-cell lymphoma and the role of other death genes in the control of apoptosis.

Apoptosis↗
PubMed1993

Programmed cell death (apoptosis) in immunity and haematological neoplasia.

Programmed cell death, also commonly referred to as apoptosis, is a genetically controlled sequence of events, often requiring protein synthesis, which results in cell death. Although initially described 20 years ago, it is only recently that its role as a mechanism in controlling cell population size and content has been fully realised. In this review the biochemical events of apoptosis are discussed briefly, followed by a more detailed look at the role of programmed cell death in the control of haemopoiesis and the maintenance of a balanced functional immune system. The role of proto-oncogenes and suppressor genes known to regulate programmed cell death is covered, and also their influence on the induction and maintenance of neoplastic disease. Finally, the potential role of apoptosis in the resistance of haematological malignancies to chemotherapy is commented on.

Animals↗
PubMedDate not supplied

Expression of HLA class II in colorectal cancer: evidence for enhanced immunogenicity of microsatellite-instability-positive tumours.

Microsatellite-instability-positive (MSI+) colorectal cancers are characterised by an accumulation of deletion and insertion mutations at simple repeated sequences caused by inactivation of mismatch repair proteins. They share several clinicopathological features, including a better prognosis and a pronounced stromal inflammatory reaction. We have used suppression subtraction hybridisation between normal colonic epithelium and paired carcinomas to generate differential gene expression profiles in MSI+ and MSI- tumours. Following reverse northern blotting analysis, 11 genes were found to be up-regulated in the MSI+ tumour, and one of these specified the HLA-DM gene. This was in contrast to the MSI(-) tumour screen, where none of the clones analysed expressed this class of gene. Our results confirm that MSI(+) tumours may have a greater potential for the efficient presentation of antigens to the helper arm of the immune system and lend further support to the theory that the better prognosis of patients with MSI+ tumours may be linked to an enhanced immunogenicity of these tumours.

Base Sequence↗
PubMedDate not supplied

Identification of differentially expressed genes associated with colorectal cancer liver metastasis.

The molecular mechanisms underlying successful metastasis of primary colorectal tumour to the liver remain unknown. We have used suppression subtractive hybridisation (SSH) and reverse Northern dot blot analysis to profile the mRNA expression patterns of a primary colorectal cancer and its liver metastasis. After SSH and reverse Northern dot blot analysis, differential expression was confirmed in 17 clones from the forward, and 13 clones from the reverse subtracted cDNA library. Four clones showed no significant sequence identities with any known sequences in the GenBank data base and likely to represent novel genes whose up- or down-regulation is associated with colorectal liver metastasis. Interestingly, one of the 13 down-regulated clones displayed 99% sequence identity with the BRCA1 tumour suppressor gene. Since promoter methylation is a direct cause of transcription silencing of the BRCA1 gene in approximately 10-20% of human breast cancer we further investigated its promoter methylation status in ten primary colorectal tumour samples, but revealed no evidence of promoter methylation.

Adult↗