Biomedical subjects

G I Murray

Publications and source records attributed to G I Murray.

At least 19 recordsLinked to original sources

PubMed2002

c-erbB-2 is not a major factor in the development of colorectal cancer.

We have investigated c-erbB-2 protein expression in a large cohort of well-characterized colorectal tumours, and in a subset of lymph node metastases. We have also evaluated a Val(655)Ile single nucleotide polymorphism, which is associated with an increased risk of breast cancer, in a subset of the colorectal cancer patients and in healthy control subjects. Immunohistochemical studies revealed that while 81.8% of tumours expressed c-erbB-2, in the majority of cases equivalent levels of c-erb-B2 were seen in adjacent normal mucosa. Colon tumours were significantly more likely to express c-erbB-2 than rectal tumours (P=0.015). Only 52.4% of the metastases displayed staining patterns concordant with their primary tumour, indicating that determination of c-erbB-2 protein in colorectal tumours cannot predict the status of lymph node metastases. PCR--RFLP analysis of the Val(655)Ile single nucleotide polymorphism demonstrated that allele frequencies were identical between colorectal cancer patients and a control group of Caucasian subjects (Ile=0.80 and Val=0.20 in each case), indicating that it is not related to the risk of developing colorectal cancer in this population. Furthermore, there was no relationship between c-erbB-2 protein expression and gene polymorphism (P=0.58). In terms of prognosis, no association was seen between either c-erbB-2 protein expression or the presence of the Val allele and patient survival (P>0.05 in each case), suggesting that c-erbB-2 is not a prognostic marker in colorectal cancer.

Adenocarcinoma↗

PubMed2002

Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases.

Overexpression of the epidermal growth factor receptor (EGFR) often correlates with an aggressive tumour phenotype and poor prognosis. To examine the relevance of EGFR in colorectal cancer, we determined the expression of EGFR protein in 249 colorectal adenocarcinomas and 42 lymph node metastases using immunohistochemistry. Moreover, we investigated a (CA)(n) dinucleotide repeat polymorphism of the EGFR gene in a subset of 114 tumours. High levels of EGFR protein were observed in 123/249 (49.4%) samples. EGFR expression in colorectal carcinomas correlated with differentiation grade (P=0.014). However, there were no associations with Dukes' stage, site, patient age or gender. EGFR protein expression did not influence survival in this colorectal cancer patient cohort (P>or=0.05). Expression was not identical in paired colorectal tumours and lymph node metastases, with only 17/42 (40.5%) samples showing equivalent EGFR levels (P>0.05). The distribution of the (CA)(n) dinucleotide repeat alleles in colorectal adenocarcinomas was not associated with EGFR protein expression (P>0.05). These results indicate that while EGFR overexpression is a common event in colorectal carcinogenesis, it does not influence patient prognosis.

Adenocarcinoma↗

PubMed2002

Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer.

AIMS: The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes collectively capable of degrading all extracellular matrix components, in particular fibrillar collagen. The importance of this group of proteins in the processes of tumour invasion and metastasis is now widely acknowledged. MMP-13 (collagenase 3) has a central role in the MMP activation cascade. The purpose of this study was to investigate the presence and activity of MMP-13 in colorectal cancer and relate these to clinicopathological features. METHODS: Immunohistochemistry for MMP-13 was performed on formalin fixed, paraffin wax embedded sections of a large series of colorectal cancers (n = 249), all of which had uniform clinical and pathological information available. Immunoreactivity to MMP-13 was detected with a monoclonal antibody to MMP-13 using a Dako TechMate 500 automated immunostaining system. The presence and cellular localisation of MMP-13 was assessed using a semiquantitative scoring system. Gelatin zymography was used to detect and measure MMP-13 activity. The zymography was performed on a subset of the cases studied by immunohistochemistry using two groups of 10 paired Dukes's C tumours and normal samples, selected by either having "good" or "poor" survival. RESULTS: Immunoreactivity to MMP-13 was identified in 91% of cases and immunoreactivity was localised to the cytoplasm of tumour cells. A high MMP-13 staining score showed a trend towards poorer survival. Tumours had significantly greater MMP-13 activity compared with normal colonic mucosa (p < 0.001). Furthermore, the tumour to normal tissue ratio was significantly higher in the poor survival group (p = 0.02). CONCLUSIONS: These results show that MMP-13 is frequently present and active in colorectal cancer and suggest that the activity of MMP-13 is associated with poorer survival in colorectal cancer.

Adult↗

PubMed2002

Tumour cytochrome P450 and drug activation.

The expression of drug metabolising cytochrome P450s (CYPs) notably 1A, 1B, 2C, 3A, 2D subfamily members have been identified in a wide range of human cancers. Individual tumour types have distinct P450 profiles as studied by detection of P450 activity, identification of immunoreactive CYP protein and detection of CYP mRNA. Selected P450s, especially CYP1B1, are overexpressed in tumours including cancers of the lung, breast, liver, gastrointestinal tract, prostate, bladder. Several prodrug anti-tumour agents have retrospectively been identified as P450 substrates for which tumour CYP activation may hitherto have been underestimated. Those in clinical use include prodrug alkylating agents (cyclophosphamide, ifosphamide, dacarbazine, procarbazine), Tegafur, a prodrug fluoropyrimidine, methoxymorphylinodoxorubicin, a metabolically activated anthracycline, as well as flutamide and tamoxifen, two non-steroidal hormone receptor antagonists that are significantly more active following CYP-hydroxylation. More exciting is the prospect of developing new agents designed to be selectively dependent on tumour CYP activation. This can be illustrated with P450 activation of the 2-(4-aminophenyl)benzothiazoles exclusively in CYP1A1 inducible tumours. Also of interest is the bioreductive antitumour prodrug AQ4N, a CYP3A substrate that is activated to a cytotoxic metabolite specifically in hypoxic tumour regions.

Antineoplastic Agents↗

PubMed2001

Colorectal cancer genomics: evidence for multiple genotypes which influence survival.

Colorectal cancer (CRC) is a leading cause of cancer death and the mechanism for variable outcome in this disease is not yet fully understood. It is hypothesized that differences in the genetic make-up of tumours may be partially responsible for the differences observed in survival among same staged individuals for this disease. In this study the tumour genomes of 29 consecutive patients undergoing surgery for Dukes' C CRC were assessed by comparative genomic hybridization (CGH). In addition, the CGH profiles from the tumours were compared with those from eight colorectal cell lines. Great variation in genetic grade (all detectable aberrations i.e., loss + gain) was observed in 29 Dukes' C colorectal tumours by CGH (median four aberrations per tumour, range 0-20). Gain was found in 76% and loss in 41% of tumours. The most frequently observed regions of gain were 13q (27.6%), 20q (27.6%), 7p (24.1%), 8q (24.1%), and 1q (20.7%) and loss were 18q (31%), 4q (20.7%), 17p (20.7%), 18p (20.7%), and 15q (20.1%). None of these specific genomic aberrations were associated with patient survival. However, patients with more than two aberrations had a better survival than patients with fewer regions of loss and gain (P = 0.02). CRC cell lines had similar regions of loss or gain as the tumours. However, the frequency of genomic aberrations was much greater in the CRC cell lines. Although genomic change in CRC is relevant to the survival of patients with Dukes' C CRC, careful analysis is required to identify cell lines which are representative models of CRC genomics.

Aged↗

PubMed2001

Thymidine phosphorylase and dihydropyrimidine dehydrogenase protein expression in colorectal cancer.

It is essential for actively proliferating cells to increase their rate of DNA synthesis to progress through the cell cycle. This is reflected in the increased uracil usage that is a common feature in solid tumours. Thymidine phosphorylase (TP) anabolises formation of pyrimidine nucleosides available for DNA synthesis, whereas dihydropyrimidine dehydrogenase (DPD) catabolises the degradation of pyrimidine bases, thereby reducing levels of uracil and thymine available for DNA synthesis. In addition, tissue levels of TP or DPD have been associated with the clinical efficacy of pyrimidine anti-metabolites commonly used in the treatment of colorectal cancer. There is little information, however, on the relative expression or degree of co-ordinated regulation of either protein in primary or metastatic colorectal cancer. DPD and TP protein levels were measured in 15 primary colorectal carcinomas, 10 colorectal liver metastases and 25 adjacent uninvolved tissues. DPD was reduced in 67% (10/15) of colorectal tumours (mean tumour/normal = 0.52) and in all liver metastases (mean tumour/normal = 0.41) compared with the corresponding normal tissue. In contrast, TP was increased in 80% (12/15) of colorectal tumours (mean tumour/normal = 18.91) and in all metastases (mean tumour/normal = 3.70). TP and DPD protein expression were highly variable in uninvolved and tumour tissues. The ratio of TP:DPD was higher in 87% of colorectal tumours and in all liver metastases compared with the adjacent uninvolved tissues. This suggests the presence of co-ordinated regulation of these pyrimidine metabolic enzymes and offers a strategy for optimising the use of pyrimidine-based chemotherapy.

Colorectal Neoplasms↗

PubMed2001

Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P = 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary.

Aryl Hydrocarbon Hydroxylases↗

PubMed2001

Cytochrome P450 CYP1B1 protein expression: a novel mechanism of anticancer drug resistance.

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.

Animals↗

PubMed2001

Matrix metalloproteinases: a multifunctional group of molecules.

The matrix metalloproteinases are a large group of zinc-containing proteases with a central role in the degradation of all types of extracellular matrix. Increased matrix degradation is a characteristic feature of several disease processes, most notably tumour invasion; it is now widely recognized that this group of proteases has a key role in facilitating invasion and metastasis. However, it is also becoming increasingly recognized that the matrix metalloproteinases are a multifunctional group of biologically important molecules with diverse roles in normal cell growth, differentiation, and cell regulation, and with involvement in the early stages of tumourigenesis. Some of these regulatory functions may be distinct from the matrix-degrading capabilities of this group of enzymes.

Breast Neoplasms↗

PubMed2001

Spot the differences: proteomics in cancer research.

Separation of thousands of cellular proteins by two-dimensional electrophoresis allows the detailed comparison of proteins from normal and diseased tissue. Mass spectrometry provides a fast and reliable way of characterising proteins of interest, particularly when the gene sequence of the source organism is known. The availability of the human genome sequence has opened up the possibility of identifying protein differences between normal and diseased tissue, thus providing the opportunity to search for tumour markers or for therapeutic targets. This new technology will give much-needed insight into the molecular mechanisms of tumour development and progression.

Humans↗

PubMed2001

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.

Animals↗

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↗