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Biomedical subjects

G I Murray

Publications and source records attributed to G I Murray.

At least 37 records · Page 2Linked to original sources
PubMed2001

Application of laser capture microdissection and proteomics in colon cancer.

AIMS: Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model. METHODS: Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel. RESULTS: Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaffected. CONCLUSION: A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed.

Colon↗
PubMed2001

Regulation, function, and tissue-specific expression of cytochrome P450 CYP1B1.

Cytochrome P450 CYP1B1 is a relatively recently identified member of the CYP1 gene family. The purpose of this commentary is to review the regulatory mechanisms, metabolic specificity, and tissue-specific expression of this cytochrome P450 and to highlight its unique properties. The regulation of CYP1B1 involves a variety of both transcriptional and post-transcriptional mechanisms. CYP1B1 can metabolize a range of toxic and carcinogenic chemicals in vitro but in some cases with a unique stereoselectivity. Estradiol 4-hydroxylation appears to be a characteristic reaction catalyzed by human CYP1B1. However, there are considerable species differences regarding the regulation, metabolic specificity, and tissue-specific expression of this P450. In humans CYP1B1 is overexpressed in tumor cells, and this has important implications for tumor development and progression and the development of anticancer drugs specifically activated by CYP1B1.

Animals↗
PubMed2001

Analysis of uracil DNA glycosylase in human colorectal cancer.

Uracil DNA glycosylase (UDG) is responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Colorectal cancer is widely treated with 5-FU, which leads to thymidylate synthase inhibition; this accounts for increased dUTP intracellular pools and subsequent uracil incorporation into DNA. Uracil misincorporation has also been implicated in the link between folate deficiency and colorectal cancer risk. As there is no information on UDG in colorectal cancer, this study characterized UDG activity and protein expression in a panel of 20 colorectal tumors and 6 colorectal cell lines. UDG activity in colorectal tissue is widely variable and it is statistically higher in tumor tissue (P=0.013) compared to normal bowel. Tumor versus normal activity ratios ranged from 0.49 to 2.2 (median 1.13). Among the six colorectal cell lines tested, UDG activity varied from 40 to 68 units and was markedly (1.7-fold) higher than in tumor tissue (P<0.0001). In both colorectal tissues and cell lines, UDG was expressed as both 29 kDa and 35 kDa forms. Total protein expression varied 3.2-fold in cell lines; variability was also found between patients and between normal and tumoral tissue for the same patient. This study demonstrates UDG protein and functional activity in human colorectal tumors and cell lines. The high tumor:normal tissue ratio supports further interest in base excision repair, through UDG, as a potential source of fluoropyrimidine resistance in colorectal cancer.

Aged↗
PubMed2001

Human CYP1B1 and anticancer agent metabolism: mechanism for tumor-specific drug inactivation?

The cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of procarcinogens and xenobiotics. Human CYP1B1 protein has been detected in a variety of tumors but is not detected in adjacent normal tissues or in liver. This suggests that CYP1B1 could biotransform anticancer agents specifically in the target cells. The interaction between CYP1B1 and 12 commonly used anticancer drugs was screened using an ethoxyresorufin deethylase assay. Four agents were competitive inhibitors of CYP1B1 activity: flutamide (K(i) = 1.0 microM), paclitaxel (K(i) = 31.6 microM), mitoxantrone (K(i) = 11.6 microM), and docetaxel (K(i) = 28.0 microM). Doxorubicin (K(i) = 2.6 microM) and daunomycin (K(i) = 2.1 microM) were mixed inhibitors, while tamoxifen was a noncompetitive inhibitor (K(i) = 5.0 microM). Vinblastine, vincristine, 5-fluorouracil, etoposide, and cyclophosphamide did not inhibit CYP1B1 activity. In vitro incubations with flutamide and CYP1B1 produced a metabolite consistent with 2-hydroxyflutamide. Comparison of kinetic parameters (K(m), K(i), V(max)) for flutamide 2-hydroxylation by CYP1B1, CYP1A1, and CYP1A2 indicate that CYP1B1 could play a major role for flutamide biotransformation in tumors. The results obtained indicate that several anticancer agents inhibit CYP1B1 activity. Drug inactivation by CYP1B1 may represent a novel mechanism of resistance, influencing the clinical outcome of chemotherapy.

Antineoplastic Agents↗
PubMed2000

Cyclin D1 protein expression and gene polymorphism in colorectal cancer. Aberdeen Colorectal Initiative.

Cyclin D1 is a key cell cycle regulatory protein, the expression and subcellular localization of which is often altered in human tumor cells. A common A/G single nucleotide polymorphism (A870G) in exon 4 of the cyclin D1 gene, CCND1, is associated with the presence of 2 distinct mRNA transcripts for this G1/S regulatory protein, and CCND1 genotype has been related to prognosis in lung cancer and head and neck carcinoma. We have investigated both the expression of cyclin D1 protein and the CCND1 A870G polymorphism in 100 colorectal cancer patients. Immunohistochemistry demonstrated cyclin D1 protein expression in 55% of tumors, and while the absence of cyclin D1 protein was not associated with outcome (p=0.81), high levels of protein expression (>50% of tumor cells expressing cyclin D1) correlated with significantly shortened overall survival (p=0.01). Using polymerase chain reaction restriction fragment length polymorphism analysis, we determined the frequency of each genotype and found that CCND1 genotype was not related to overall survival (p>0.05). In addition, genotype was unrelated to the level of expression and localization of cyclin D1 protein, as well as other key G1/S checkpoint proteins (p21, p27, p53, retinoblastoma) and tumor proliferation markers (proliferating cell nuclear antigen). However, higher levels of p27, and to a lesser extent p21, were associated with reduced cytoplasmic cyclin D1 protein (p=0.029 and p=0.054, respectively). In conclusion, we have demonstrated that high levels of cyclin D1 protein expression are related to outcome in colorectal cancer; however, the CCND1 A870G polymorphism is unrelated to either cyclin D1 protein expression or patient survival.

Adenocarcinoma↗
PubMed2000

Genomic instability at the BUB1 locus in colorectal cancer, but not in non-small cell lung cancer.

Genomic instability is observed in the majority of human tumors. Dysregulation of the mitotic spindle checkpoint is thought to be one of the mechanisms that facilitate aneuploidy in tumor cells. Mutations in the mitotic spindle checkpoint kinase BLUB1 cause a dominant negative disruption of the spindle, leading to chromosome instability in cancer cell lines. However, little is known about chromosome 2q14, the genomic region containing BUB1, in human tumors. The BUB1 locus was evaluated in 32 colorectal cancer (CRC) and 20 non-small cell lung cancer (NSCLC) primary tumors using a panel of seven microsatellite repeats for 2q, two CA repeats in BUB1, and gene mutation analysis. The 2q locus was allelically stable in NSCLC but relatively unstable in colorectal primary tumors (20 of 32 tumors, 62.5%). In addition, 14.5% of CRC patients displayed instability within BUB1. Previously described BUB1 mutations and polymorphisms were rare (< 1%) in the CRC or NSCLC tumors. Our data demonstrate 2q and BUB1 allelic instability in CRC and indicate that mutations in BUB1 are rare causes of chromosome instability in CRC or NSCLC. Additional investigations may shed light on the mechanistic impact of the mitotic spindle checkpoint pathway in colorectal tumor initiation and progression.

Adenocarcinoma↗
PubMed2000

The role of cytochrome P450 in tumour development and progression and its potential in therapy.

The cytochromes P450 (P450) are a large group of constitutive and inducible haem-containing enzymes, which have a central role in the oxidative metabolism of a diverse range of xenobiotics. Many P450 substrates are carcinogenic, while other substrates are anti-cancer drugs; the P450s thus have various potentially important roles in tumour biology. Several P450 genes are polymorphic and are associated with the increased risk of cancer development in specific tissues. Individual P450s, especially CYP1B1, are overexpressed in different types of tumours. The increased expression of P450s in tumours is highly significant and is important for understanding tumour development and progression. The tumour-specific expression of P450 provides the basis for the development of novel diagnostic and therapeutic strategies.

Animals↗
PubMed2000

Proteomics: a new approach to the study of disease.

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins.

Biomarkers↗
PubMed2000

Matrix metalloproteinases: molecular aspects of their roles in tumour invasion and metastasis.

The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes, whose physiological functions include tissue remodelling and embryogenesis. The importance of this group of proteins in the processes of tumour invasion and metastasis is now widely acknowledged, and has led to the search for MMP inhibitors for use as anticancer treatments in a clinical setting. This review aims to bring the reader up-to-date with current research relating to MMPs, with particular emphasis on emerging mechanisms of regulation of these enzymes, and their interaction with cell adhesion molecules. The therapeutic inhibition of MMPs will also be discussed.

Cadherins↗
PubMed2000

Laser capture microscopy.

Human tissues are composed of complex admixtures of different cell types and their biologically meaningful analysis necessitates the procurement of pure samples of the cells of interest. Many approaches have been used in attempts to overcome this difficulty, including a variety of microdissection methods. This review concerns a recent advance in microdissection techniques, namely laser capture microdissection (LCM). The principle underlying this technique is outlined, and practical issues pertaining to LCM are considered. In addition, the literature relating to LCM is reviewed, with examples of research applications of this technique being outlined.

Cell Separation↗
PubMed2000

Application of the enrichment approach to identify putative markers of response to 5-fluorouracil therapy in advanced colorectal carcinomas.

A wide range of tumor response is seen amongst patients with the same stage of colorectal cancer, even with the use of uniform chemotherapy. The significant economic and personal impact of chemotherapy provides the impetus for the identification of markers of response for use in guiding patient treatment. However, practical constraints prevent evaluation of all putative markers in a definitive manner. In this study, the enrichment approach was evaluated by examining the expression of a panel of putative response markers in selected patient populations with advanced colorectal cancer (i.e., those demonstrating the best and the poorest clinical response to a standardized 5-fluorouracil/folinic acid chemotherapy regimen). Patients showing a good response had a significantly increased survival when compared with poor responders (P=0.0013). Markers were then ranked for clinical importance based on differences in expression between the two groups. This allows for the relatively rapid and inexpensive investigation of multiple markers, using defined patient groups. Bcl-2 overexpression in primary colorectal tumor specimens was found to correlate with clinical response of metastatic deposits to chemotherapy (P=0.044), as did the site of the primary tumor (P=0.011). However, no clear association was observed between response status and the other examined factors (p53, PCNA, TP, MMPs 1, 2 or 9, TIMPs 1 or 2, TS, Dukes' stage at initial diagnosis, histological grade, sex or age). This approach has allowed prioritization of markers of clinical response on which larger, statistically definitive studies will be performed.

Adult↗
PubMed2000

Expression of cell cycle control proteins in primary colorectal tumors does not always predict expression in lymph node metastases.

Analysis of tumor markers focuses on expression in primary tumors with the assumption that this is representative of metastatic tumor, against which treatment is targeted. Few studies have compared the expression of such markers in primary and secondary tumors. In this study, several key genes involved in cell cycle regulation were investigated in colorectal tumors and corresponding lymph node metastases. The cell cycle regulators p53, cyclin D1, p21, p27, retinoblastoma protein (Rb), and proliferating cell nuclear antigen (PCNA) were examined in a series of 42 paired samples of primary colorectal and secondary lymph node tumors by immunohistochemistry. Expression of p53, p27, and Rb was similar in virtually all paired samples (p53, 38 of 42; p27, 39 of 42; Rb, 40 of 42), indicating that the pattern of these proteins in colorectal tumors may be used to predict that in lymph node tumors. It also suggests a lack of direct involvement in the metastatic process. A lower concordance for p21 and cyclin D1 staining was observed between primary and secondary tumors (p21, 19 of 42; cyclin D1, 22 of 42). p21 expression was more often observed in primary colorectal cancers, whereas cyclin D1 expression was more frequently seen in lymph node metastases, in keeping with the contrasting roles of these proteins as a cell cycle inhibitor (p21) and activator (cyclin D1). The PCNA-labeling index was found to vary considerably in a number of cases, thus limiting the ability to predict expression of this protein in lymph node metastases from the primary tumor. In addition, PCNA-labeling indices between paired samples were neither consistently higher nor lower, suggesting that the proliferative capacity of tumor cells is not directly related to their ability to metastasize.

Adult↗
PubMed1999

Differential expression of CYP1A1, CYP1A2, CYP1B1 in human kidney tumours.

The presence of mRNA of individual members of the CYP1 gene family in normal and neoplastic kidney has been investigated by RTPCR. CYP1B1 mRNA was consistently expressed in both normal and neoplastic kidney while CYP1A1 was present in the majority of normal and neoplastic whereas CYP1A2 was infrequently expressed. Expression of the Ah receptor and Arnt which are involved in the transcriptional activation of the CYP1 genes was also studied. The Ah receptor mRNA and Arnt mRNA were consistently expressed both in kidney tumours and normal kidney. These results indicate differential expression of individual members of the CYP1 gene family in normal and neoplastic kidney and suggest that several mechanisms including the Ah receptor complex could be involved in their regulation.

Actins↗
PubMed1999

Matrix metalloproteinases in tumour invasion and metastasis.

The matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes, which are involved in the degradation of many different components of the extracellular matrix. The MMPs have been classified into different groups including collagenases, gelatinases, stromelysins, and others, particularly membrane-type MMPs, based mainly on the in vitro substrate specificity of individual MMPs. There is increasing evidence to indicate that individual MMPs have important roles in tumour invasion and metastasis. However, the current concept of the role of MMPs in tumour invasion is that they not only have a direct role in tumour invasion by facilitating extracellular matrix degradation, but as a consequence they also have an important role in maintaining the tumour micro-environment and thus promoting tumour growth. Inhibiting the action of MMPs represents a new therapeutic approach for the treatment of individual types of cancer and several broad-spectrum, low-molecular-weight MMP inhibitors are currently being assessed for clinical use. This review examines the role of MMPs in tumour invasion and metastasis, with an emphasis on studies of clinical relevance.

Humans↗
PubMed1999

Cytochrome P450 CYP3A in human renal cell cancer.

Renal cell cancer is the main malignant tumour of the kidney and has an increasing incidence. This type of tumour has a poor prognosis and shows intrinsic resistance to several anti-cancer drugs. The CYP3A P450 family, which consists of three closely related forms, is involved in the oxidative activation and deactivation of a variety of carcinogens and several anti-cancer drugs. In this study the presence and cellular localization of CYP3A has been investigated using a combination of immunohistochemistry, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR) in renal cell cancer and corresponding normal kidney. CYP3A was consistently expressed in both renal call cancer and in normal kidney. In renal cell cancer, CYP3A was localized to tumour cells and in normal kidney the predominant cellular localization of CYP3A was to proximal tubular epithelial cells. RT-PCR showed that both CYP3A5 mRNA and CYP3A7 mRNA were consistently present in both tumour and normal samples, while CYP3A4 mRNA was present in 65% of tumours and 90% of normal samples. This study indicates that individual members of the CYP3A family are expressed in renal cell cancer. The presence of CYP3A in renal cell cancer might be important in the metabolic potentiation as well as the detoxification of chemotherapeutic agents used to renal cancer.

Adult↗