Sir Peter MacCallum Department of Oncology - Research Publications
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ItemNo Preview AvailableA DOMINANT-NEGATIVE MUTANT OF MSOS1 INHIBITS INSULIN-INDUCED RAS ACTIVATION AND REVEALS RAS-DEPENDENT AND RAS-INDEPENDENT INSULIN SIGNALING PATHWAYS(AMER SOC MICROBIOLOGY, 1995-01)The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing either wild-type (CHO-IR/SOS cells) or mutant (CHO-IR/delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.
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ItemNo Preview AvailableTissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice(AMER SOC MICROBIOLOGY, 1998-08)The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.
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ItemA phase III study of radiotherapy with and without continuous-infusion fluorouracil as palliation for non-small-cell lung cancer(NATURE PUBLISHING GROUP, 1997)This study assesses the effect of adding continuous-infusion fluorouracil to palliative thoracic radiation therapy (RT) on the rate and duration of symptom relief in patients with advanced non-small-cell lung cancer (NSCLC). Two hundred eligible patients with NSCLC were randomized to receive either 20 Gy in five daily fractions as palliation for intrathoracic disease or the same RT with concurrent continuous infusion of 1 g m(-2) day(-1) fluorouracil for 5 days. Survival, response and rates of symptom relief in the two groups were compared according to treatment intent, and toxicities were compared according to treatment received. The overall response rate was higher in patients randomized to the combination (29%) than in patients randomized to RT alone (16%) (P = 0.035). However, there were no significant differences between the treatment arms in terms of overall or progression-free survival or in palliation of symptoms. Patients treated with RT plus fluorouracil had significantly more acute toxicity, including nausea and vomiting (P = 0.01), oesophagitis (P = 0.0003), stomatitis (P = 0.0005) and skin reaction (P = 0.003). This study suggests for the first time an interaction between RT and infusional fluorouracil in NSCLC. Although RT plus fluorouracil resulted in a significantly higher response rate than achieved with RT alone, this did not translate into more effective palliation. Because the combination produced significantly more toxicity than RT alone, it is not recommended for the palliative treatment of NSCLC. Nevertheless, these results suggest that opportunities may exist for exploitation of the observed enhancement of antitumour effect in the setting of high-dose radical RT for NSCLC.
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ItemOrmaplatin resistance is associated with decreased accumulation of its platinum (II) analogue, dichloro(D,L-trans)1,2-diaminocyclohexaneplatinum (II).(Springer Science and Business Media LLC, 1996-08)Ormaplatin (also known as tetraplatin) is a platinum-containing analogue which has recently undergone clinical trials. Ormaplatin may undergo conversion to dichloro(D,L-trans)-1,2-diaminocyclohexaneplatinum(II) [P1Cl2(trans-dach)]. The cisplatin-resistant murine lymphoma cell lines E8 and E5, were found to be cross-resistant to ormaplatin and PtCl2(trans-dach). We found an inverse rank correlation between drug resistance and drug accumulation for PtCl2(trans-dach) similar to our previous findings with cisplatin; however, accumulation of ormaplatin in the resistant cells was increased. Ormaplatin cytotoxicity appears to result primarily from extracellular conversion to PtCl2(trans-dach), since ormaplatin cytotoxicity was decreased under conditions where extracellular conversion to PtCl2(trans-dach) was minimised. Co-incubation with different inhibitors of energy metabolism resulted in a 65-70% increase in PtCl2(trans-dach) accumulation in the parental cell line R1.1 and a 113-307% increase in the resistant cell line E5 which suggests that the decrease in accumulation in E5 may be at least partly energy dependent. We conclude from these findings that cross-resistance to ormaplatin is associated with an energy-dependent decreased accumulation of PtCl2(trans-dach) in these cisplatin-resistant cell lines.
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ItemTP53 intron 6 polymorphism and the risk of ovarian and breast cancer(CHURCHILL LIVINGSTONE, 1998-02)
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ItemBRCA1 polymorphisms(CHURCHILL LIVINGSTONE, 1997)
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ItemLOSS OF HETEROZYGOSITY AND AMPLIFICATION ON CHROMOSOME-11Q IN HUMAN OVARIAN-CANCER(STOCKTON PRESS, 1993-02)The 11q13 chromosomal region encodes oncogenes relevant to a variety of human cancers as well as a tumour suppressor gene implicated in multiple endocrine neoplasia type 1. In addition, high affinity folate receptor (FOLR1), which maps to 11q13.3-13.5, is expressed at an elevated level on the surface of over 80% of nonmucinous epithelial ovarian cancers. Further telomeric, 11q breakpoints are found in many cancers. We studied the involvement of 11q markers in ovarian cancer by looking for tumour-specific loss of heterozygosity (LOH), as well as amplification or rearrangements that might explain the overexpression of FOLR1. Twenty eight epithelial ovarian cancers, along with lymphocyte DNA from the same individual were used for Southern blotting with polymorphic probes from 11q. PCR primers from 11q23.3 were also used. The 11q13 band was amplified in four out of 28 cancers. The amplicon included the probe D11S146 as well as FGF3 (formerly INT2) and FOLR1 in one out of these four cases, thus crossing the bcl1 translocation breakpoint. LOH was seen in three out of 16 cases with FGF3 (11q13). A much higher frequency of LOH (8/12) was found at 11q23.3-qter, implying the presence of a tumour suppressor gene in this region.
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ItemMDM2 OVEREXPRESSION IS RARE IN OVARIAN-CARCINOMA IRRESPECTIVE OF TP53 MUTATION STATUS(STOCKTON PRESS, 1995-10)Somatic mutations in TP53 are seen in many human cancers. In addition, the protein product of the wild-type TP53 can be sequestered by the protein MDM2 (murine double minute 2). This protein is commonly overexpressed in human sarcomas and gliomas, usually as a result of gene amplification. In this study, 43 ovarian carcinomas (OCs) were analysed for aberrations in the TP53 gene by immunohistochemistry (IHC), loss of heterozygosity (LOH) or mutation analysis. The MDM2 gene and its product was studied by Southern blotting and IHC. Over 50% of the OCs studied showed mutations in TP53 by either direct sequencing (19/36, 53%), positive IHC (23,43, 53%) or both, whereas 0/32 had amplification of MDM2 and only 1/37 tumours had positive IHC using the anti-MDM2 antibody IF-2. The solitary example of positive IHC in this series was seen in a mixed müllerian tumour with sarcomatous differentiation and was not accompanied by MDM2 DNA amplification. These results support previous data showing that around 50% of OCs have mutations in TP53 and in addition, suggest that MDM2 is not amplified in OC, but the presence of sarcomatous features in mixed müllerian tumours may result in positive immunohistochemistry with IF-2.
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ItemAllele loss and mutation screen at the Peutz-Jeghers (LKB1) locus (19p13.3) in sporadic ovarian tumours(CHURCHILL LIVINGSTONE, 1999-04)Germline mutations in the LKB1 (STK11) gene (chromosome sub-band 19p13.3) cause characteristic hamartomas and pigmentation to develop in patients with Peutz-Jeghers syndrome. Peutz-Jeghers syndrome carries an overall risk of cancer that may be up to 20 times that of the general population and Peutz-Jeghers patients are at increased risk of benign and malignant ovarian tumours, particularly granulosa cell tumours. Loss of heterozygosity (allele loss, LOH) has been reported in about 50% of ovarian cancers on 19p13.3. LKB1 is therefore a candidate tumour suppressor gene for sporadic ovarian tumours. We found allele loss at the marker D19S886 (19p13.3) in 12 of 49 (24%) sporadic ovarian adenocarcinomas. Using SSCP analysis, we screened ten ovarian cancers with LOH, 35 other ovarian cancers and 12 granulosa cell tumours of the ovary for somatic mutations in LKB1. No variants were detected in any of the adenocarcinomas. Two mutations were detected in one of the granulosa cell tumours: a mis-sense mutation affecting the putative 'start' codon (ATG --> ACG, M1T); and a silent change in exon 7 (CTT --> CTA, leucine). Like BRCA1 and BRCA2, therefore, it appears that LKB1 mutations can cause ovarian tumours when present in the germline, but occur rarely in the soma. The allele loss on 19p13.3 in ovarian cancers almost certainly targets a different gene from LKB1.
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ItemBRCA1 mutations in southern England(CHURCHILL LIVINGSTONE, 1998-06)If genetic testing for breast and ovarian cancer predisposition is to become available within a public health care system there needs to be a rational and cost-effective approach to mutation analysis. We have screened for BRCA1 mutations in 230 women with breast cancer, all from the Wessex region of southern England, in order to establish the parameters on which to base a cost-effective regional mutation analysis strategy. Truncating mutations were detected in 10/155 (6.5%) consecutive cases selected only for diagnosis under the age of 40 (nine of these ten women had a strong family history of breast or ovarian cancer), 3/61 (4.9%) bilateral-breast cancer cases (all three mutations occurring among women for whom the first cancer was diagnosed under 40 years) and 8/30 (26.6%) breast cancer cases presenting to the genetics clinic (for whom a strong family history of breast and/or ovarian cancer was present). Ten different mutations were detected in 17 families, but three of these accounted for 10/17 (59%) of the families. The cost of screening the population for mutations in the entire BRCA1 gene is unacceptably high. However, the cost of screening a carefully selected patient cohort is low, the risk of misinterpretation much less and the potential clinical benefits clearer.