• Lapatinib versus hormone therapy in patients with advanced renal cell carcinoma: a randomized phase III clinical trial.

      Ravaud, Alain; Hawkins, Robert E; Gardner, Jason P; Von der Maase, Hans; Zantl, Niko; Harper, P; Rolland, Frédéric; Audhuy, Bruno; Machiels, Jean-Pascal; Pétavy, Frank; et al. (2008-05-10)
      PURPOSE: Lapatinib is an orally reversible inhibitor of epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER-2) tyrosine kinases with demonstrated activity in patients with HER-2-positive breast cancer. In the current phase III open-label trial, lapatinib was compared with hormone therapy (HT) in patients with advanced renal cell carcinoma (RCC) that express EGFR and/or HER-2. PATIENTS AND METHODS: Patients with advanced RCC who had experienced disease progression through first-line cytokine therapy--stratified by Karnofsky performance status and number of metastatic sites--were randomly assigned to lapatinib 1,250 mg daily or HT. The primary end point was time to progression (TTP); secondary end points included overall survival (OS), safety, and biomarker analyses. RESULTS: Four hundred sixteen patients were enrolled onto the study. Median TTP was 15.3 weeks for lapatinib versus 15.4 weeks for HT (hazard ratio [HR] = 0.94; P = .60), and median OS was 46.9 weeks for lapatinib versus 43.1 weeks for HT (HR = 0.88; P = .29). In a biomarker analysis of patients with EGFR-overexpressed tumors (3+ by immunohistochemistry [IHC]; n = 241) median TTP was 15.1 weeks for lapatinib versus 10.9 weeks for HT (HR = 0.76; P = .06), and median OS was 46.0 weeks for lapatinib versus 37.9 weeks for HT (HR = 0.69; P = .02). These results were confirmed by Cox regression analysis. No unexpected toxicities were observed; the most commonly reported drug-related adverse events (all grades) for lapatinib were rash (44%) and diarrhea (40%). CONCLUSION: Lapatinib was well tolerated with equivalent overall efficacy to HT in advanced RCC patients who had experienced disease progression while receiving cytokines, and the study supports that lapatinib prolonged OS relative to HT in patients with 3+ EGFR status determined by IHC.
    • Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

      Johnson, Suzanne M; Banyard, Antonia; Smith, Christopher; Mironov, A.; McCabe, Martin; Children's Cancer Group, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology Medicine and Health, University of Manchester, Oglesby Cancer Research Building, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, Manchester M20 4GJ, UK. (2020)
      Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.
    • The Large Scale Synthesis and Chromatographic and Spectral Properties of a Series of Alkylated Thymine and Uracil-Containing Nucleosides. O2-, 3- and O4-Alkylpyrimidine Derivatives

      Saffhill, Roy; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom (1987)
    • Large-scale immunoprecipitation from fission yeast cell extracts.

      Grallert, Agnes; Hagan, Iain M; CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom (2017-02-01)
      We outline immunoprecipitation (IP) procedures to isolate the large quantities of a molecule of interest that are required to identify posttranslational modifications (PTMs) in subsequent targeted mass spectrometry analysis. In situ denaturation by trichloroacetic acid precipitation inhibits the activities of modifying enzymes that could alter the PTM profile to preserve the PTMs on a target of interest throughout the precipitation step. In contrast, isolation of the same molecule with the nondenaturing variation on this IP procedure can maintain associations with partner molecules whose PTMs can also be mapped, albeit with the caveat that modifications could have occurred during the extended IP period.
    • Laser flash photolysis of rhodopsin at room temperature

      Bensasson, R V; Land, Edward J; Truscott, T G (2011-08-08)
    • Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection.

      Weekes, M; Tan, S; Poole, E; Talbot, S; Antrobus, R; Smith, Duncan L; Montag, C; Gygi, S; Sinclair, J; Lehner, P; et al. (2013-04-12)
      The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance-associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14(+) and CD34(+) progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.
    • Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA.

      Stacey, Simon N; Jordan, Deborah; Williamson, Andrew J K; Brown, Michael D; Coote, Joanna H; Arrand, John R; Cancer Research Campaign, Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX, United Kingdom. sstacey@picr.man.ac.uk (2000-08)
      Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.
    • Less G(2) arrest in irradiated cells of breast cancer patients than in female controls: a contribution to their enhanced chromosomal radiosensitivity?

      Scott, David; Spreadborough, Anne R; Roberts, Stephen A; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. dscott@picr.man.ac.uk (2003-06)
      PURPOSE: To determine if the efficacy of G(2) checkpoint control (measured as the degree of mitotic inhibition) was reduced in breast cancer patients (n=129) compared with healthy controls (n=105) after exposure of lymphocytes to X-rays. We had previously shown that the average level of radiation-induced chromosome damage was higher in G(2) lymphocytes of these patients than in the controls, and it was proposed that this was a marker of low penetrance predisposition to cancer. MATERIALS AND METHODS: Proliferating lymphocytes were X-irradiated (50 cGy) and sampled at 90 min post-irradiation, which was the time of maximum mitotic inhibition of G(2) cells, expressed as the extent of reduction in the mitotic index in irradiated compared with unirradiated cells. RESULTS: Repeated measurements on 28 controls showed that there were reproducible differences in mitotic inhibition between individuals. Inhibition was significantly greater in female than in male controls (p=0.014), but less in patients than in female controls (p=0.009). There was a weak inverse correlation between the extent of inhibition and the amount of chromosome damage in all females (r=-0.15, p=0.043). CONCLUSIONS: The lesser mitotic inhibition in patients than in female controls might contribute to their greater mean G(2) chromosomal radiosensitivity. However, this hypothesis is not easily reconciled with other observations that (1) the significant difference in inhibition between the sexes in controls was not accompanied by any gender difference in radiosensitivity and (2) there was an inverse correlation between inhibition and age in controls, yet no age-related increase in radiosensitivity. There might, therefore, be no causal relationship between G(2) mitotic inhibition and chromosomal radiosensitivity.
    • Letter to the editor in response to 'When to apply sunscreen: a consensus statement for Australia and New Zealand'

      Baldwin, L; Olsen, CM; Gordon, L; Green, Adèle C; Aitken, J; Neale, R; Whiteman, D; Janda, M; Institute of Health and Biomedical Research, School of Public Health and Social Work, Faculty of Health, Queensland University of Technology, Australia (2019)
    • Leukocyte migration inhibition in human pulmonary neoplasia.

      Vose, Brent M; Kimber, I; Moore, Michael; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1977-03)
      Peripheral blood leukocytes from 65 lung cancer patients, 69 healthy donors, 33 patients with malignant disease outside the lung, and 24 patients with nonmalignant pulmonary conditions were examined for immune reactivity, as measured by the leukocyte migration inhibition assay, to antigens in homogenates of lung tumor tissue and material derived from tumor-free lung areas and a nonmalignant lung lesion. A low level of reactivity with apparent specificity was detected in the lung cancer patient group, with 17/52 showing migration inhibition with extracts of lung tumor tissue. Reactivity was also detectable in this group against homogenates of tumor-free lung and nonmalignant lung lesion. The data supported the conclusion that sensitization at least in some patients with lung cancer might be directed not only against tumor-specific antigens but also against antigens of less confined disease association.
    • Levels of the DNA adduct, N7-methyldeoxyguanosine, are associated with increased risk of failure of treatment of cervical intraepithelial neoplasia.

      Acladious, N N; Harrison, Kathryn L; Sutton, C J; Povey, Andrew C; Mandal, D; Kitchener, Henry C; Department of Genito-Urinary Medicine, Manchester Royal Infirmary, Manchester, UK. (2004-06)
      OBJECTIVE: To determine whether exposure to methylating agents was a risk factor for treatment failure in women undergoing colposcopic examination. METHODS: Nine hundred fifty-eight women attending for colposcopic examination after abnormal cervical smear test results were recruited into the study cohort. Information on demographic factors, smoking and other risk factors was obtained and a pre-treatment biopsy was taken and stored at -70 degrees C. After follow-up, cases who had treatment failure of cervical intraepithelial neoplasia (CIN) within 2 years following treatment were identified (n = 77) and matched to women with no treatment failure of CIN in this time period (controls, n = 154). DNA was extracted from the pre-treatment biopsies and levels of N7-methyl-deoxyguanosine (N7-MedG), a marker of exposure to methylating agents, were quantified as the ring-opened form of the base damage by a validated immunoslotblot assay. RESULTS: Sufficient DNA for N7-MedG analysis was extracted from 61 subjects corresponding to 20 matched case control pairs. N7-MedG was detected in cervical DNA with levels ranging from non-detected (<0.1 micromol/mol dG) to 4.83 micromol/mol dG. N7-MedG levels were significantly higher in cases (geometric mean 0.99 micromol/mol dG) than controls (0.33 micromol/mol dG; P = 0.01). There were no associations between N7-MedG levels and HPV or smoking status. Log N7-MedG content, after adjustment for HPV status at time of treatment, was found to be significantly associated with increased risk of treatment failure (OR 5.74, 95% CI 1.05-31.23). CONCLUSIONS: The association between pre-treatment levels of DNA damage induced by methylating agents and subsequent treatment failure implicates methylating agent exposure as a causative factor in treatment failure.
    • Li-Fraumeni syndrome--a molecular and clinical review.

      Varley, Jennifer; Evans, D Gareth R; Birch, Jillian M; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, UK. (1997)
    • Light and scanning electron microscopy of the same human metaphase chromosomes.

      Harrison, Christine J; Jack, E; Allen, Terence D; Harris, R; Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester, M20 9BX, UK. (1985-08)
      A technique has been developed to examine the same G-banded human metaphase chromosomes, first in the light microscope and then in the scanning electron microscope (SEM). A structural involvement in chromosome banding was confirmed by a positional correlation between the G-positive bands observed in the light microscope and the circumferential grooves between the quaternary coils of the metaphase chromosomes, observed in the SEM. In further support of this the regions between the grooves showed a positional relationship with the G-negative or reverse (R) bands. The examination of slightly extended metaphase chromosomes in the light microscope demonstrated that the G-banding pattern corresponded to that described by the Paris nomenclature for metaphase chromosomes. The arrangement of the circumferential grooves of the same chromosomes, observed in the SEM, was shown to relate to that described by the Paris nomenclature for prometaphase chromosomes. Therefore, using the SEM it is possible to demonstrate the details of prometaphase banding in metaphase chromosomes.
    • Limitations of immune complex measurements in colorectal disease.

      Hobbiss, J; Cooper, K M; Moore, Michael; Gowland, E; Schofield, P F; Department of Surgery, Univerity Hospital of South Manchester, Withington, Manchester (1983-08)
      Three techniques (Clq, Raji and L1210 binding assays) alleged to measure circulating immune complexes (ICs) were applied to the sera of 101 patients with colorectal disease (54 carcinoma; 23 inflammatory; 13 benign tumour and 11 miscellaneous) at the time of diagnostic or definitive surgery, and 58 healthy adult controls. Elevated levels in the pathological sera were observed by all 3 methods in order of sensitivity: Raji greater than Clq greater than L1210. However, none of them differentiated between benign, inflammatory and neoplastic conditions nor, in the case of colorectal carcinoma, was there any correlation with stage of disease. With the exception of Raji v. L1210 (r = 0.43, P less than 0.001), correlations between the various assays were poor and levels of serum carcinoembryonic antigen (CEA) did not correlate with ICs measured by any of the techniques. Indeed, the IC assays were even less discriminatory than CEA, which was elevated mainly in the serum of carcinoma patients and which was positively correlated with serum gamma-glutamyl transpeptidase (gamma GT) (r = 0.42, P less than 0.005). The data suggest that the lack of concordance between the IC assays is a reflection of heterogeneity among ICs, interfering factors present in pathological sera, or both. Thus the IC assays deployed here have neither diagnostic nor prognostic utility in colorectal disease at this time, and immunochemical characterization of the serum reactive material detected by the different assays is required.
    • The lineage commitment of haemopoietic progenitor cells.

      Cross, Michael A; Enver, T; Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, UK. exhmc@picr.cr.man.ac.uk (1997-10)
      Multipotent haemopoietic progenitor cells appear to be 'primed' for commitment by co-expression of a multiplicity of genes characteristic of different lineages. Lineage commitment proceeds as the consolidation of a distinct pattern of gene expression out of this milieu.
    • Lineage restriction of the RARalpha gene expression in myeloid differentiation.

      Zhu, Jun; Heyworth, Clare M; Glasow, Annegret; Huang, Qiu-Hua; Petrie, Kevin; Lanotte, Michael; Benoit, Gerard; Gallagher, Robert; Waxman, Samuel; Enver, Tariq; et al. (2001-10-15)
      To better understand the role of retinoids in myelopoiesis, expression of the retinoid receptor genes (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) were examined during differentiation of factor-dependent cell-Paterson (FDCP)-mixA4 murine progenitor cells. The major receptor expressed in undifferentiated A4 cells was RARalpha (primarily the RARalpha1 isoform). Following induction of myelomonocytic differentiation with granulocyte and granulocyte-macrophage colony-stimulating factors, a dramatic increase in RARalpha expression (particularly the RARalpha2 isoform) was seen. In contrast, expression of both RARalpha isoforms was rapidly extinguished upon induction of erythroid differentiation with erythropoeitin (EPO). A modest induction of RXRalpha expression was seen, particularly during differentiation in the myelomonocytic lineage. Low expression levels of RARgamma2 and RXRbeta remained unchanged, irrespective of differentiation pathway. Consistent with the gene expression patterns, RARalpha agonists and antagonists stimulated myelomonocytic and erythroid differentiation of FDCP-mixA4 cells, respectively. Taken together, these results suggest that erythropoiesis and granulopoiesis require diminished and enhanced RARalpha activities, respectively, which at physiological all-trans-retinoic acid (RA) concentrations may be accomplished by reciprocal effects of EPO and myelomonocytic growth factors on its expression. This hypothesis is corroborated by data showing that RA, which positively regulates RARalpha2 expression, can exert inhibitory effects on erythroid differentiation.
    • Linkage studies in a Li-Fraumeni family with increased expression of p53 protein but no germline mutation in p53.

      Birch, Jillian M; Heighway, Jim; Teare, M Dawn; Kelsey, Anna M; Hartley, Ann L; Tricker, K J; Crowther, Derek; Lane, D P; Santibanez-Koref, Mauro F; University of Manchester, CRC Paediatric & Familial Cancer Research Group, Christie Hospital, UK. (1994-12)
      We report a family with the Li-Fraumeni syndrome (LFS) in whom we have been unable to detect a mutation in the coding sequence of the p53 gene. Analysis of linkage to three polymorphic markers within p53 enabled direct involvement of p53 to be excluded. This is the first example of a LFS family in whom exclusion of p53 has been possible. Four affected members of the family with sarcoma or premenopausal breast cancer showed increased expression of p53 protein in their normal tissues as detected by immunohistochemistry. It therefore appears that the LFS phenotype has been conferred by an aberrant gene, showing a dominant pattern of inheritance, which may be acting to compromise normal p53 function rather than by a mutation in p53 itself. In order to try to determine the chromosomal location of this putative gene, we have carried out studies of linkage to candidate loci. By these means we have excluded involvement of Rb1 and BRCA1 on chromosomes 13q and 17q respectively. The MDM2 oncogene on chromosome 12q was considered to be the prime candidate as MDM2 is amplified in sarcomas and the MDM2 product binds to p53. Furthermore, p53 mutation and amplification of MDM2 have been shown to be mutually exclusive events in tumour development. Linkage analysis to two polymorphic markers within MDM2 yielded a three-point LOD score of -5.4 at a recombination fraction theta equal to zero. Therefore MDM2 could be excluded. It is possible that the gene which is responsible for cancer susceptibility in this family, possibly via interaction with p53, will be important in the histogenesis of breast cancer in general. We are now carrying out further studies to locate and identify this gene.
    • A linkage study in seven breast cancer families.

      Teare, M Dawn; Santibanez-Koref, Mauro F; Wallace, S A; White, Gavin R M; Evans, D Gareth R; Burnell, Liza D; Harris, Martin; Howell, Anthony; Birch, Jillian M; Cancer Research Campaign Pediatric and Familial Cancer Research Group, Christie Hospital, Manchester, England. (1993-04)
      Seven breast cancer families are examined for evidence of linkage to a site in the region of 17q12-q21, by using five markers. The families constitute a subset of a larger series of familial breast cancer; the seven families were selected because constitutional DNA was available on informative members, either from clinical samples or extracted from paraffin blocks. Two-point lod scores are reported. The maximum lod score, 0.8824, is obtained with marker NM23 at theta = 0. This is clearly not significant in itself; however, when taken in context with evidence from existing reports, it provides support for linkage to this region.
    • Linked parallel synthesis and MTT bioassay screening of substituted chalcones.

      Lawrence, Nicholas J; Rennison, David; McGown, Alan T; Ducki, Sylvie W; Gul, Lubna A; Hadfield, John A; Khan, Nader; Department of Chemistry, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester, M60 1QD, UK. Lawrencenj1@cardiff.ac.uk (2001)
      A 644-membered library of chalcones was prepared by parallel synthesis using the Claisen-Schmidt base-catalyzed aldol condensation of substituted acetophenones and benzaldehydes. The cytotoxicity of these chalcones was conveniently determined upon the crude products directly in 96-well microtiter test plates by the conventional MTT assay. This method revealed seven chalcones of IC(50) less than 1 microM of which 4'-hydroxy-2,4,6,3'-tetramethoxychalcone (5a) was the most active [IC(50) (K562), 30 nM]; it causes cell cycle arrest at the G(2)/M point and binds to tubulin at the colchicine binding site.
    • The lipid fluidity of rat liver membrane subfractions.

      Whetton, Anthony D; Houslay, M D; Dodd, Nicholas J F; Evans, W H; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX (1983-09-15)
      1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.r. techniques. 2. The sinusoidal (blood-facing) plasma-membrane subfraction was the most fluid of the three plasma-membrane regions. Fractions originating from the bile-canalicular and contiguous (lateral) regions were most rigid. Endocytic fractions isolated (endosomes and diacytosomes) were of a similar fluidity to fractions originating from the sinusoidal plasma-membrane region. By far the most fluid fractions examined were derived from the Golgi-apparatus complex. 3. The three plasma-membrane subfractions each showed a different response to the bilayer-fluidizing effect of benzyl alcohol. 4. Arrhenius-type plots of the order parameter S and outer hyperfine splitting, 2T( parallel), identified lipid-phase separations in the plasma-membrane subfractions.