• The M-CSF receptor substrate and interacting protein FMIP is governed in its subcellular localization by protein kinase C-mediated phosphorylation, and thereby potentiates M-CSF-mediated differentiation.

      Mancini, Annalisa; Koch, Alexandra; Whetton, Anthony D; Tamura, Teruko; Institut für Biochemie, OE 4310, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. (2004-08-26)
      Macrophage colony-stimulating factor (M-CSF or CSF-1) and its cognate receptor, the tyrosine kinase c-fms, are essential for monocyte and macrophage development. We have recently identified an Fms-interacting protein (FMIP) that binds transiently to the cytoplasmic domain of activated Fms molecules and is phosphorylated on tyrosine by Fms tyrosine kinase. FMIP is a substrate not only for Fms but also for protein kinase C (PKC). Mutagenesis reveals that this occurs on serines 5 and 6. Adjacent to these sites is a nuclear localization signal (NLS). We show that this NLS is essential for the predominantly nuclear localization of FMIP. Generation of phosphomimetic substitutions on serine residues 5 and 6 confirms that PKC-mediated phosphorylation on this site leads to translocation of FMIP to the cytosol. Furthermore, the mutant FMIP (FMIPSS5,6AA) was detected abundantly in the nucleus even in the presence of activated PKCalpha. Wild-type FMIP and FMIPSS5,6AA inhibited M-CSF-mediated survival signaling, while FMIPSS5,6EE-expressing cells survived and differentiated into macrophages more efficiently than wild-type cells in the presence of M-CSF or TPA. We conclude M-CSF-mediated activation of PKCalpha can potentiate FMIP action to initiate survival/differentiation signaling.
    • Machine learning and data mining frameworks for predicting drug response in cancer: an overview and a novel in silico screening process based on association rule mining

      Vougas, K; Sakellaropoulos, T; Kotsinas, A; Foukas, GP; Ntargaras, A; Koinis, F; Polyzos, A; Myrianthopoulos, V; Zhou, H; Narang, S; et al. (2019)
      A major challenge in cancer treatment is predicting the clinical response to anti-cancer drugs on a personalized basis. The success of such a task largely depends on the ability to develop computational resources that integrate big "omic" data into effective drug-response models. Machine learning is both an expanding and an evolving computational field that holds promise to cover such needs. Here we provide a focused overview of: 1) the various supervised and unsupervised algorithms used specifically in drug response prediction applications, 2) the strategies employed to develop these algorithms into applicable models, 3) data resources that are fed into these frameworks and 4) pitfalls and challenges to maximize model performance. In this context we also describe a novel in silico screening process, based on Association Rule Mining, for identifying genes as candidate drivers of drug response and compare it with relevant data mining frameworks, for which we generated a web application freely available at: https://compbio.nyumc.org/drugs/. This pipeline explores with high efficiency large sample-spaces, while is able to detect low frequency events and evaluate statistical significance even in the multidimensional space, presenting the results in the form of easily interpretable rules. We conclude with future prospects and challenges of applying machine learning based drug response prediction in precision medicine.
    • Machine learning approaches on high throughput NGS data to unveil mechanisms of function in biology and disease

      Pezoulas, V. C.; Hazapis, O.; Lagopati, N.; Exarchos, T. P.; Goules, A. V.; Tzioufas, A. G.; Fotiadis, D. I.; Stratis, I. G.; Yannacopoulos, A. N.; Gorgoulis, Vassilis G; et al. (2021)
      In this review, the fundamental basis of machine learning (ML) and data mining (DM) are summarized together with the techniques for distilling knowledge from state-of-the-art omics experiments. This includes an introduction to the basic mathematical principles of unsupervised/supervised learning methods, dimensionality reduction techniques, deep neural networks architectures and the applications of these in bioinformatics. Several case studies under evaluation mainly involve next generation sequencing (NGS) experiments, like deciphering gene expression from total and single cell (scRNA-seq) analysis; for the latter, a description of all recent artificial intelligence (AI) methods for the investigation of cell sub-types, biomarkers and imputation techniques are described. Other areas of interest where various ML schemes have been investigated are for providing information regarding transcription factors (TF) binding sites, chromatin organization patterns and RNA binding proteins (RBPs), while analyses on RNA sequence and structure as well as 3D dimensional protein structure predictions with the use of ML are described. Furthermore, we summarize the recent methods of using ML in clinical oncology, when taking into consideration the current omics data with pharmacogenomics to determine personalized treatments. With this review we wish to provide the scientific community with a thorough investigation of main novel ML applications which take into consideration the latest achievements in genomics, thus, unraveling the fundamental mechanisms of biology towards the understanding and cure of diseases.
    • Machine learning approaches to improve prostate cancer outcome

      Pereira, Ronnie R; Miller, Crispin J; Baena, Esther; Bristow, Robert G; The University of Manchester, Manchester (2019)
    • Machine learning based models of radiotherapy-induced skin induration for breast cancer patients

      Cicchetti, A.; La Rocca, E.; De Santis, M. C.; Seibold, P.; Azria, D.; De Ruysscher, D.; Valdagni, R.; Dunning, A. M.; Elliot, R.; Gutierrez-Enriquez, S.; et al. (2022)
      Purpose or Objective To use data from an international prospective cohort study of breast cancer patients (pts) to predict the risk of skin induration (SI) after radiotherapy (RT) using a machine learning algorithm that includes dosimetric/clinical/genetic factors. Materials and Methods Pts were treated after breast conserving surgery with conventional/moderate or ultra hypo-fractionated RT with or without a tumour bed boost based on clinical and pathological factors. Pts were enrolled in 7 countries in Europe/US; each centre followed local clinical practice, but the collection of data and genotyping was standardised and centralised. Our endpoint was late grade 1+ (G1+) SI 2 years after RT completion. Inclusion criteria were: no SI at baseline and availability of complete dosimetric and genetic data. For every pt, skin was defined as a 5-mm inner isotropic expansion from the outer body. To select a relevant portion of the skin DVH, we extracted the higher dose tail using different volume cutoffs (i.e. 25/50/100/150/200 cc volumes corresponding to 5x5-20x20cm2 areas). We corrected sub-DHVs for fractionation using two possible a/b values from the literature (1.8 Gy, Bentzen 1988 & Raza 2012; 3.6 Gy, Jones 2006 & Budach 2015). We calculated Equivalent Uniform Doses (EUDs) from corrected sub-DVHs, with n spanning from 1 to 0.05. We also considered the minimum dose of the selected DVH tail as an additional dose parameter (Dmin). Toxicity models were built using feed-forward neural networks (FNNs, 10 neurons and 1 hidden layer) following a wrapper method for feature selection. We used separate datasets for input: clinical/treatment/genetic features were constant, while the dosimetric factors (EUDs and Dmin) coming from sub-DVHs varied with volume cutoff and a/b .Results The 647 pts included in the analysis had a G1+ SI rate at 2 years of 29.4%. 281 variables were considered: 127 published SNPs (GWAS literature), 40 clinical factors, 93 treatment factors and 21 dosimetric variables (for each volume and a/b). For volume thresholds <200cc, no dosimetric feature was selected by the wrapper method. Therefore, we derived a predictive model (16 features, no dosimetric variable) for use before RT planning (Model 1). At sub-DVH_200cc, for a/b=3.6Gy only Dmin was selected (Model 2) as dosimetric variable, while for a/b=1.8Gy, EUD (n=0.5) and Dmin entered the FNN (Model 3). Conclusion A pre-planning SI model was derived that included information on genetics (6 SNPs), treatment (6 RT, 1 oncology) and clinical factors. Largest volume (200cc) sub-DVH allowed selection of dosimetric features, particularly with a/b=1.8Gy and EUD with n=0.5. Following validation, the model could be used to personalise use of new RT schedules, such as ultrahigh hypofractionation, to minimise risk of Sl.
    • Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy.

      Allen, Terence D; Bennion, G R; Rutherford, S A; Reipert, Siegfried; Ramalho, A; Kiseleva, Elena; Goldberg, Martin W; CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K. (1997-09)
      Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM "working resolution." Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance.
    • Macrophage inflammatory protein 1alpha attenuates the toxic effects of temozolomide in human bone marrow granulocyte-macrophage colony-forming cells.

      Clemons, Mark; Watson, Amanda J; Howell, Anthony; Chang, James; Heyworth, Clare M; Lord, Brian I; Testa, Nydia G; Dexter, T Michael; Margison, Geoffrey P; Cancer Research Campaign Department of Medical Oncology, Manchester, United Kingdom. markclemons@sprint.ca (2000-03)
      Macrophage inflammatory protein 1alpha (MIP-1alpha) is a chemokine that may act principally by preventing hemopoietic cells from entering G1, thereby attenuating the cytotoxic effects of cell cycle-specific chemotherapeutic agents. Here we examine the effect of MIP-1alpha on the sensitivity of human granulocyte-macrophage hemopoietic progenitor cells (granulocyte-macrophage colony-forming cells; GM-CFCs) with the cytotoxic effects of antitumor agents that act mainly via alkylation at the O6 position of guanine in DNA. Mononuclear cell preparations from human bone marrow were used in an in vitro GM-CFC colony-forming assay. The GM-CFC survival from individual patients displayed a range of sensitivities to the methylating agent temozolomide [(Tz) 20-55% survival at 10 microg/ml Tz]. However, in all 16 cases, MIP-1alpha (50 ng/ml) protected against GM-CFC killing: survival in the presence of MIP-1alpha ranged from 65-97% at 10 microg/ml Tz, with GM-CFCs being 1.5-4.5-fold more resistant than control cells from the same patient. The highest levels of protection were seen in the GM-CFCs with the highest sensitivity in the absence of MIP-1alpha. Similar degrees of protection were seen for the methylating agent streptozotocin, but no protection was detected for the chloroethylating agents carmustine or mitozolomide in the samples for which there was protection against the toxic effects of Tz. Whereas the mechanism of this effect remains to be established, the results may have potential immediate clinical application in the attenuation of hematological toxicity after administration of methylating antitumor agents.
    • Macrophage inflammatory protein-1 alpha mediated growth inhibition in a haemopoietic stem cell line is associated with inositol 1,4,5 triphosphate generation.

      Heyworth, Clare M; Pearson, Mark A; Dexter, T Michael; Wark, G; Owen-Lynch, P J; Whetton, Anthony D; Department of Experimental Haematology, Paterson Institute, Manchester, UK. (1995)
      Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.
    • Macrophage inflammatory protein: its characteristics, biological properties and role in the regulation of haemopoiesis.

      Lord, Brian I; Heyworth, Clare M; Woolford, Lorna B; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1993-06)
      Studies on haemopoietic stem cells had led to the realisation that negative feedback inhibitors play an important role in regulating their proliferation. One such molecule was identified as MIP-1 alpha. One of a family of cytokines, originally recognised as inflammatory molecules, MIP-1 alpha is now potentially valuable as a means of manipulating and protecting haemopoietic (and possibly other) stem cells during chemotherapy. This short review briefly considers the structural classification of MIP-1 alpha and its molecular relatives and indicates some of the probable human/murine equivalent molecules outlining the evidence for the equivalence of MIP-1 alpha (murine) and LD78 (human). Sources of MIP-1 alpha/LD78 are identified as monocyte/macrophage and lymphocytic cells and their role in inflammatory responses is seen to be significant. All proliferation in haemopoietic tissue is now recognised as a major target for MIP-1 alpha action. In vitro it synergises with certain growth factors to promote progenitor cell colony formation, but effects are dependent on the maturational age of the cells promoted. With more primitive cells it is seen as inhibitory. This property is particularly valuable in vivo where MIP-1 alpha can protect stem cells against the effects of cytotoxic agents. Since it appears that leukaemic stem cell proliferation is not inhibited, MIP-1 alpha/LD78 present great potential for stem cell protection in the theatre of cytotoxic therapies.
    • Macrophage-inflammatory protein protects multipotent hematopoietic cells from the cytotoxic effects of hydroxyurea in vivo.

      Lord, Brian I; Dexter, T Michael; Clements, J M; Hunter, M A; Gearing, A J; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1992-05-15)
      Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has been assessed for its potential in vivo to protect hematopoietic progenitor cells from the cytotoxic effects of a cycle-specific drug--in this case hydroxyurea (HU). Two doses of HU, 7 hours apart, were administered to mice to induce spleen colony-forming unit (CFU-S) cycling and then to kill them during DNA-synthesis. MIP-1 alpha, in a variety of dose and time combinations, was injected before the second dose of HU in an attempt to prevent recruitment or maintain CFU-S quiescence, and thus protect them from the second dose of HU. Without MIP-1 alpha, recovery of the CFU-S population was complete in 7 days. In a dose-dependent manner, MIP-1 alpha either reduced the initial kill and accelerated recovery, or completely protected the CFU-S population. We conclude that MIP-1 alpha does protect multipotent progenitor cells in vivo and that these observations provide a base from which to build practical clinical applications.
    • MaeIII polymorphism in sequence encoding 3' untranslated region of the MCC gene.

      Heighway, Jim; Hoban, Paul R; Wyllie, A H; CRC Department of Cancer Genetics, Paterson Institute of Cancer Research, Manchester, UK. (1992-03-11)
    • The Magnolia Trial: Zanubrutinib, a next-generation bruton tyrosine kinase inhibitor, demonstrates safety and efficacy in relapsed/refractory marginal zone lymphoma

      Opat, S.; Tedeschi, A.; Linton, Kim M; McKay, P.; Hu, B.; Chan, H.; Jin, J.; Sobieraj-Teague, M.; Zinzani, P. L.; Coleman, M.; et al. (2021)
      Purpose: Marginal zone lymphoma (MZL) is an uncommon non-Hodgkin lymphoma with malignant cells that exhibit a consistent dependency on B-cell receptor signaling. We evaluated the efficacy and safety of zanubrutinib, a next-generation selective Bruton tyrosine kinase inhibitor, in patients with relapsed/refractory (R/R) MZL. Experimental design: Patients with R/R MZL were enrolled in the phase 2 MAGNOLIA (BGB-3111-214) study. The primary endpoint was overall response rate (ORR) as determined by an independent review committee (IRC) based on the Lugano 2014 classification. Results: Sixty-eight patients were enrolled. After a median follow-up of 15.7 months (range, 1.6 to 21.9 months), the IRC-assessed ORR was 68.2% and complete response (CR) was 25.8%. The ORR by investigator assessment was 74.2%, and the CR rate was 25.8%. The median duration of response (DOR) and median progression-free survival (PFS) by independent review was not reached. The IRC-assessed DOR rate at 12 months was 93.0%, and IRC-assessed PFS rate was 82.5% at both 12 and 15 months.with the majority of adverse events (AEs) being grade 1 or 2. The most common AEs were diarrhea (22.1%), contusion (20.6%), and constipation (14.7%). Atrial fibrillation/flutter was reported in two patients; one patient had grade 3 hypertension. No patient experienced major hemorrhage. In total, four patients discontinued treatment due to AEs, of which was considered treatment-related by the investigators. Conclusions: Zanubrutinib demonstrated high ORR and CR rate with durable disease control and a favorable safety profile in patients with R/R MZL.
    • Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium.

      Booth, Catherine; O'Shea, Julie A; Potten, Christopher S; Epithelial Biology Group, CRC Section of Cell and Tumour Biology, Paterson Institute, Christie Hospital (NHS) Trust, Manchester, M20 4BX, United Kingdom. CBooth@picr.man.ac.uk (1999-06-15)
      We have previously described a method for the primary culture of adult large intestinal epithelium, suggesting that stem cells had survived both the isolation and the culture procedures. However, as no markers for such cells exist, confirmation of stem cell survival is difficult-only the functional properties can be used to define them. Unfortunately, many of these (e.g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epithelium which gradually became multicellular and formed invaginations containing many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt-like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical crypt-like morphology. Hence, functional adult stem cells appear to survive isolation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell characterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy.
    • The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway.

      Gros, Laurent; Ishchenko, Alexander A; Ide, Hiroshi; Elder, Rhoderick H; Saparbaev, Murat K; Groupe Réparation de l'ADN', UMR 8113 CNRS, LBPA-ENS Cachan, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. (2004)
      In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.
    • Making connections at DNA replication forks: Mrc1 takes the lead.

      Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-10-24)
      In a recent issue of Molecular Cell, Lou et al. (2008) demonstrate that the Mrc1 protein associates with the DNA polymerase that acts on the leading strand at replication forks, suggesting a potential mechanism that could help to preserve genome stability.
    • Making connections: p53 and the cathepsin proteases as co-regulators of cancer and apoptosis

      Soond, S. M.; Savvateeva, L. V.; Makarov, V. A.; Gorokhovets, N. V.; Townsend, Paul A; Zamyatnin, A. A., Jr.; Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow, Russia. (2020)
      While viewed as the "guardian of the genome", the importance of the tumor suppressor p53 protein has increasingly gained ever more recognition in modulating additional modes of action related to cell death. Slowly but surely, its importance has evolved from a mutated genetic locus heavily implicated in a wide array of cancer types to modulating lysosomal-mediated cell death either directly or indirectly through the transcriptional regulation of the key signal transduction pathway intermediates involved in this. As an important step in determining the fate of cells in response to cytotoxicity or during stress response, lysosomal-mediated cell death has also become strongly interwoven with the key components that give the lysosome functionality in the form of the cathepsin proteases. While a number of articles have been published highlighting the independent input of p53 or cathepsins to cellular homeostasis and disease progression, one key area that warrants further focus is the regulatory relationship that p53 and its isoforms share with such proteases in regulating lysosomal-mediated cell death. Herein, we review recent developments that have shaped this relationship and highlight key areas that need further exploration to aid novel therapeutic design and intervention strategies.
    • Male caregivers of patients with breast and gynecologic cancer: experiences from caring for their spouses and partners.

      Lopez, Violeta; Copp, Gina; Molassiotis, Alexander; Research Centre for Nursing and Midwifery Practice, Medical School, Australian National University, Canberra, Australia. violeta.lopez@anu.edu.au (2012)
      There is considerable evidence demonstrating the negative effects of caregiving particularly in the areas of psychological well-being and quality of life of family caregivers of patients with cancer. However, there is little work on male caregivers' subjective experience of caring for family members with cancer, and little is known on how caregivers experience the caring over time.
    • Malignant myelomonocytic cells after in vitro infection of marrow cells with Friend leukaemia virus.

      Testa, Nydia G; Dexter, T Michael; Scott, David; Teich, N M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1980-01)
      Infection of long-term BDF1 marrow cultures with Friend leukaemia virus complex (FLV) induced transformed cells with myelomonocytic characteristics, which were isolated only 14 days after the viral infection. Criteria for transformation were growth in suspension cultures and high plating efficiency in agar. The lymphatic leukaemia virus (LLV) replicates in these suspension cultures, but the spleen focus-forming virus (SFFV) component of the FLV complex has not been detected. Injection of the transformed cells into syngeneic neonatal or adult mice leads to the development of leukaemia which can be demonstrated to be of donor origin by the presence of two metacentric marker chromosomes which are also seen in the cultured cells.
    • Malignant transformation and genetic alterations are uncoupled in early colorectal cancer progression

      Mamlouk, S.; Simon, T.; Tomás, L.; Wedge, David C; Arnold, A.; Menne, A.; Horst, D.; Capper, D.; Morkel, M.; Posada, D.; et al. (2020)
      Background: Colorectal cancer (CRC) development is generally accepted as a sequential process, with genetic mutations determining phenotypic tumor progression. However, matching genetic profiles with histological transition requires the analyses of temporal samples from the same patient at key stages of progression. Results: Here, we compared the genetic profiles of 34 early carcinomas with their respective adenomatous precursors to assess timing and heterogeneity of driver alterations accompanying the switch from benign adenoma to malignant carcinoma. In almost half of the cases, driver mutations specific to the carcinoma stage were not observed. In samples where carcinoma-specific alterations were present, TP53 mutations and chromosome 20 copy gains commonly accompanied the switch from adenomatous tissue to carcinoma. Remarkably, 40% and 50% of high-grade adenomas shared TP53 mutations and chromosome 20 gains, respectively, with their matched carcinomas. In addition, multi-regional analyses revealed greater heterogeneity of driver mutations in adenomas compared to their matched carcinomas. Conclusion: Genetic alterations in TP53 and chromosome 20 occur at the earliest histological stage in colorectal carcinomas (pTis and pT1). However, high-grade adenomas can share these alterations despite their histological distinction. Based on the well-defined sequence of CRC development, we suggest that the timing of genetic changes during neoplastic progression is frequently uncoupled from histological progression.
    • Mammalian cells expressing Escherichia coli O6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes.

      Abril, N; Margison, Geoffrey P; CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester M20 4BX, U.K. (1999-06)
      The effect of expression of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, on the growth inhibitory effects of the dibromoalkanes (DBA) dibromomethane (DBM) and dibromoethane (DBE) was determined in Chinese hamster lung fibroblasts transfected with and expressing high levels of the Escherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E. coli ada gene encoding either O6-alkylguanine (O6-alkG) or alkylphosphotriester (alkPT) ATase activities. The functional activity of the ATase in these cells was demonstrated by in vitro assay of cell extracts using 3H-methylated DNA as a substrate, and by the protection they provided against the growth inhibitory effects of methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) and the chloroethylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU). However, cells expressing the full length or the O6-alkG ATase region, but not the alkPT ATase region, of Ada were found to be more sensitive to the growth inhibitory effects of the DBA; Ogt expression sensitized cells to DBM but not significantly to DBE. Addition of DBA to cell extracts depleted O6-alkG ATase activity on the methylated DNA substrate, but had no effect on alkPT ATase activity. This suggests that ATase-mediated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthionine sulfoximine in attempts to augment or deplete cellular levels of GSH had no marked effect on the ATase-mediated sensitization to DBA. This suggests that rather than GSH-mediated DNA damage, the effect may be mediated by a DNA adduct caused by the oxidative metabolic pathway. These observations indicate that expression of ATase may have a detrimental effect on cellular sensitivity to environmentally relevant alkylating agents.