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Biomedical subjects

P G Johnston

Publications and source records attributed to P G Johnston.

At least 19 recordsLinked to original sources
PubMed2006

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Base Sequence↗
PubMed2006

Pharmacoeconomic analysis of adjuvant oral capecitabine vs intravenous 5-FU/LV in Dukes' C colon cancer: the X-ACT trial.

Oral capecitabine (Xeloda) is an effective drug with favourable safety in adjuvant and metastatic colorectal cancer. Oxaliplatin-based therapy is becoming standard for Dukes' C colon cancer in patients suitable for combination therapy, but is not yet approved by the UK National Institute for Health and Clinical Excellence (NICE) in the adjuvant setting. Adjuvant capecitabine is at least as effective as 5-fluorouracil/leucovorin (5-FU/LV), with significant superiority in relapse-free survival and a trend towards improved disease-free and overall survival. We assessed the cost-effectiveness of adjuvant capecitabine from payer (UK National Health Service (NHS)) and societal perspectives. We used clinical trial data and published sources to estimate incremental direct and societal costs and gains in quality-adjusted life months (QALMs). Acquisition costs were higher for capecitabine than 5-FU/LV, but higher 5-FU/LV administration costs resulted in 57% lower chemotherapy costs for capecitabine. Capecitabine vs 5-FU/LV-associated adverse events required fewer medications and hospitalisations (cost savings pound3653). Societal costs, including patient travel/time costs, were reduced by >75% with capecitabine vs 5-FU/LV (cost savings pound1318), with lifetime gain in QALMs of 9 months. Medical resource utilisation is significantly decreased with capecitabine vs 5-FU/LV, with cost savings to the NHS and society. Capecitabine is also projected to increase life expectancy vs 5-FU/LV. Cost savings and better outcomes make capecitabine a preferred adjuvant therapy for Dukes' C colon cancer. This pharmacoeconomic analysis strongly supports replacing 5-FU/LV with capecitabine in the adjuvant treatment of colon cancer in the UK.

Administration, Oral↗
PubMed2006

c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIP(L) and c-FLIP(S) with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53(+/+), RKO), null (HCT116p53(-/-)) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIP(L), but not c-FLIP(S), potently inhibited apoptosis induced by chemotherapy in HCT116p53(+/+) cells, suggesting that c-FLIP(L) was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIP(L) synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAIL-independent, DR5- and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

Antineoplastic Agents↗
PubMed2006

Prognostic significance of glycoprotein pMQ1 in breast cancer.

BACKGROUND: A novel glycoprotein, pMQ1, is positively correlated with increasing histological grade in malignant astrocytomas. Cerebral metastases from breast cancer have also been found to contain pMQ1-positive cells. This study aimed to determine the role of pMQ1 in primary breast cancer. METHODS: Breast cancer specimens were analysed for pMQ1 by immunohistochemistry. The expression of pMQ1 was correlated with conventional prognostic indicators. Kaplan-Meier analyses were performed to compare clinical outcome between pMQ1-positive and pMQ1-negative tumours. RESULTS: pMQ1 was expressed in most of the breast cancer specimens. The surrounding normal tissue margins and benign breast tissues always lacked pMQ1 expression. A significant positive correlation was observed between pMQ1 expression and histological grade, the presence of lymphovascular invasion and Nottingham Prognostic Index. Cancers that were pMQ1 positive were significantly more likely to develop a local recurrence. CONCLUSION: pMQ1 appears to be a tumour-associated protein. The positive correlation of pMQ1 with histological grade, presence of lymphovascular invasion and Nottingham Prognostic Index suggests that it confers an adverse prognosis.

Adult↗
PubMed2005

Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients.

Folate is required for DNA synthesis, repair and methylation. Low folate status has been implicated in carcinogenesis, possibly as a result of higher rate of genetic damage. The aim of this study is to compare folate status and levels of DNA damage between breast cancer and benign breast disease control patients. Fasting blood samples from 64 histologically confirmed untreated breast cancer patients (mean age 57 years) and 30 benign breast disease control patients (mean age 51 years) were obtained. Red cell folate (RCF) and plasma homocysteine were measured. Mononuclear cells (MNC) were isolated for genetic damage analysis using the basic alkaline comet assay. Results are expressed as tail moment. Data were log transformed as appropriate before analysis for normalisation purposes. The geometric mean (95% confidence interval) of RCF (ng ml(-1)) in breast cancer patients was 339.07 (333.3-404.6) vs 379.5 (335.8-505.2) in control patients (P = 0.24). Corresponding plasma homocysteine concentrations (micromol l(-1)) were 11.9 (10.6-16.4) vs 10.1 (9.3-11.9) (P = 0.073), respectively. The mean tail moment (s.d.) of DNA damage in MNC of breast cancer patients detected by the basic comet assay was 1.4 (0.66) vs -0.17 (0.79) in controls (P < 0.0001, t-test), the modified comet assay 'endonuclease III (Endo III)' was 1.7 (0.70) vs 0.86 (0.81) (P < 0.0001, t-test), and the modified comet assay 'formamidopyrimidine glycosylase (FPG)' was 1.6 (0.62) vs 0.99 (0.94) (P < 0.0001, t-test). There was a significant negative correlation between RCF levels and DNA damage detected by modified comet assay 'FPG' (Pearson Correlation Coefficient r2 = -0.26, P = 0.02) and DNA damage was found to be significantly higher in MNC of breast cancer patients compared to benign breast disease control patients. Breast cancer patients tended to have lower RCF levels and higher levels of plasma homocysteine, but these differences were not significant. The study provides preliminary evidence that reduced folate status may be implicated in the aetiology of breast cancer perhaps by increasing the in vivo level of genetic instability.

Alcohol Drinking↗
PubMed2005

Molecular mechanisms of drug resistance.

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Tumours may be intrinsically drug-resistant or develop resistance to chemotherapy during treatment. Acquired resistance is a particular problem, as tumours not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different mechanisms of action. Resistance to chemotherapy is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumour cells may also reduce the effectiveness of chemotherapy in the adjuvant setting. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanisms of drug resistance that operate to reduce drug sensitivity in cancer cells. Drug resistance can occur at many levels, including increased drug efflux, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis. Advances in DNA microarray and proteomic technology, and the ongoing development of new targeted therapies have opened up new opportunities to combat drug resistance. We are now able to characterize the signalling pathways involved in regulating tumour cell response to chemotherapy more completely than ever before. This will facilitate the future development of rational combined chemotherapy regimens, in which the newer targeted therapies are used in combination with cytotoxic drugs to enhance chemotherapy activity. The ability to predict response to chemotherapy and to modulate this response with targeted therapies will permit selection of the best treatment for individual patients.

Antineoplastic Agents↗
PubMed2004

Mapping the distribution of the telomeric sequence (T2AG3)n in the 2n = 14 ancestral marsupial complement and in the macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization.

In this study we test the theory that the presence of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) at the centromeres of Australian marsupial 2n = 14 complements is evidence that these karyotypes are recently derived, which is contrary to the generally held view that the 2n = 14 karyotype is ancestral for Australasian and American marsupials. Here we compare the distribution of the (T(2)AG(3))( n ) sequence and constitutive heterochromatin in the presumed ancestral 2n = 14 complement and in complements with known rearrangements. We found that where there were moderate to large amounts of constitutive heterochromatin, the distribution of the (T(2)AG(3))(n) sequence reflected its presence as a native component of satellite DNA rather than its involvement in past rearrangements. The presence of centromeric heterochromatin in all Australian 2n = 14 complements therefore suggests that centromeric sites of the (T(2)AG(3))(n) sequence do not represent evidence for recent rearrangements.

Animals↗
PubMed2003

Cancer mortality in Ireland, 1926-1995.

BACKGROUND: Investigation of long time series of cancer data can still be very useful in helping to identify Cancer Control priorities and achievements. Since the partition of Ireland into the independent Republic of Ireland and Northern Ireland, which remained part of the United Kingdom, cancer mortality data have been published in an essentially similar format in both countries. The information presented here will contribute to providing a basis for the collaborative Cancer Research programme initiated recently. PATIENTS AND METHODS: Cancer mortality data have been assembled and analysed separately for the Republic of Ireland and Northern Ireland: the data have then been combined to present mortality rates for the whole of Ireland, covering the period from 1926 to 1995. Several rubrics had to be aggregated to provide data continuously over the time span (e.g. colon and rectum and cervix and body of the uterus). When data were only available in 10-year classes of age, the EM algorithm was employed to obtain 5-year age-specific rates. All rates presented are age-standardised, employing the World Standard Population. RESULTS: In women, the death rate from all neoplasms combined increased very slightly from 117 per 100 000 in 1946-1950 to 120 per 100 000 in 1991-1995. In men, the death rate increased from 127 per 100 000 to 172 per 100 000 over the same time period. The overall cancer death rate in Ireland is currently similar to the European average in men, although in women it is among the top fifth of national cancer mortality rates in European countries. While cancer is a major cause of death in Ireland, there is no evidence of an evolving epidemic building up: the death rates from most forms of cancer are declining towards the end of the time period considered. CONCLUSIONS: As demonstrated by falling death rates from Hodgkin's disease and testicular cancer, major treatment advances appear to have been incorporated effectively into clinical practice in Ireland. Progress is apparent in tobacco control and further initiatives in this area must be undertaken since tobacco appears to be the only major new carcinogen introduced recently into the Irish environment during the period covered by this study. Effective population-based screening programmes for cervix and breast cancer and, more controversially, consideration of a National Prostate Cancer Screening programme, offer scope for further improvement in mortality. Examination of this long time series of mortality data from Ireland provides information about the evolving cancer pattern and provides the necessary background to evaluate the impact of the cross-border cancer research activities now being launched.

Adult↗
PubMed2002

Trends in bacteraemia on the haematology and oncology units of a UK tertiary referral hospital.

As part of ongoing surveillance of infection in the haematology and oncology units at Belfast City Hospital, microbiologically documented bloodstream infections over three 12-month periods 1994/5, 1998/9 and 1999/00 were reviewed. Gram-positive organisms were the most common cause of blood stream infection in the haematology unit causing 66%, 56% and 64% of episodes of monomicrobial bacteraemia in 1994/5, 1998/9 and 1999/00, respectively. In haematology patients, enterococci have emerged as an important cause of bacteraemia, with increasing levels of glycopeptide resistance, and the 'non-fermenting Gram-negative rods other than Pseudomonas aeruginosa' are an increasingly common cause of monomicrobial and polymicrobial bacteraemia. In oncology patients, Gram-negative organisms (predominantly enterobacteriaceae) were more common than Gram-positive organisms, causing 50% and 54% of monomicrobial bacteraemia in 1998/9 and 1999/00, respectively. Changes in patient population, underlying diseases and chemotherapeutic agents may explain these findings. The spectrum of infection seen in haematology and oncology patients changes as management evolves. Ongoing co-operation between haematologists, oncologists and microbiologists is important to detect trends in epidemiology, which can be used to design empirical antibiotic regimens and guide infection control policies.

Adolescent↗
PubMed2002

Mapping the distribution of the telomeric sequence (T(2)AG(3))(n) in rock wallabies, Petrogale (Marsupialia: Macropodidae), by fluorescence in situ hybridization. ii. the lateralis complex.

The distribution of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) was examined by fluorescence in situ hybridization in the six Petrogale (rock wallabies) taxa of the lateralis complex. As expected, the (T(2)AG(3))(n) sequence was located at the termini of all chromosomes in all taxa. However, the sequence was also present at several nontelomeric (viz., interstitial and centromeric) sites. The signals identified were associated with either ancient rearrangements involved with the formation of the 2n = 22 plesiomorphic macropodine karyotype or more recent rearrangements associated with karyotypes derived from the 2n = 22 karyotype. Interstitial (T(2)AG(3))(n) signals identified on chromosomes 3 and 4 in all six species of the lateralis complex and a large centromeric signal identified on chromosome 7 in the five subspecies/races of P. lateralis appear to be related to the more ancient rearrangements. Subsequent chromosome evolution has seen these signals retained, lost, or amplified in different Petrogale lineages. Within the lateralis complex, in two submetacentric chromosome derived by recent centric fusions, the telomeric sequence was identified at or near the centromere, indicating its retention during the fusion process. In the two taxa where chromosome 3 was rearranged via a recent centromeric transposition to become an acrocentric chromosome, the telomeric signal was located interstitially.

Animals↗
PubMed2002

Sex chromosome elimination, X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae).

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.

Animals↗
PubMed2001

Nuclear thymidylate synthase expression, p53 expression and 5FU response in colorectal carcinoma.

Thymidylate synthase (TS) is a key enzyme in DNA synthesis and is inhibited by metabolites of the chemotherapeutic agent 5-fluorouracil (5FU). Nuclear expression of TS in human tissue in vivo has not been characterised and its clinicopathological correlates in malignancy are unknown. 52 cases of primary colorectal carcinoma (CRC) and 24 cases of matched metastatic carcinoma were studied immunohistochemically using the monoclonal antibody TS106. The degree of nuclear TS immunostaining correlated closely with levels of TS mRNA expression amongst 10 CRCs studied. Strong nuclear immunostaining was seen in normal basal crypt colonocytes and germinal centre cells, and in a varying proportion of adenocarcinoma cells. Amongst the primary carcinomas, higher TS nuclear expression was associated with prominent extracellular mucin production and right-sided location. Higher TS nuclear expression also showed a significant association with poorer response to protracted venous infusional 5FU therapy. There was no clear association between TS nuclear expression and Ki67 or p53 expression assessed immunohistochemically. There was a strong positive correlation between TS nuclear expression in primary and metastatic CRC but the latter generally showed higher expression than matched primary tumour tissue. These findings confirm the nuclear expression of TS protein in human cells in vivo and provide new insight into how such expression may relate to the behaviour of CRCs.

Adenocarcinoma↗
PubMed2001

BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents.

BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an effect that was preceded by an acute arrest at the G2/M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adriamycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these effects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds.

Antineoplastic Agents↗
PubMed2001

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3.

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

Alternative Splicing↗
PubMed2001

P53 mutation does not affect prognosis in ovarian epithelial malignancies.

Mutations of the p53 tumour suppressor gene have been found in most human cancers, including ovarian epithelial malignancies. This study investigated whether the presence or absence of p53 mutation was associated with outcome following platinum-based chemotherapy in patients with ovarian cancer. DNA samples from tumour tissue and blood were obtained from 73 patients with primary tumours, 50 of whom received platinum-based adjuvant chemotherapy. Single-strand conformation polymorphism analysis and direct DNA sequencing of exons 5-8 detected mutations in 44% (32 of 73) of tumours. These were more common in late-stage (III or IV) than in early-stage disease (I or II) (p=0.03). There was no association with histological type, volume of residual disease following surgery, or initial CA125 levels. No significant association was found between p53 status and overall survival or disease-free survival following chemotherapy. Likewise, there was no correlation between p53 mutation and response to chemotherapy as defined by normalization of CA125 levels. Tumours with p53 missense mutations recurred within a significantly shorter time than those with normal p53 (p=0.04). In addition, there was a tendency for tumours with missense mutations to have a shorter disease-free survival than those with non-missense mutations, although this did not reach statistical significance (p=0.07).

Biomarkers, Tumor↗