• 06-Methyldeoxyguanosine in oesophageal dna among individuals at high risk of oesophageal cancer

      Umbenhauer, D; Wild, C; Montesano, R; Saffhill, Roy; Boyle, John M; Huh, N; Kirstein, U; Thomale, Jürgen; Rajewsky, M; Lu, S; et al. (1985)
    • (1)H, (13)C, (15)N backbone resonance assignment for the 1-164 construct of human XRCC4

      Cabello-Lobato, Maria J; Schmidt, Christine K; Cliff, M. J.; Manchester Cancer Research Centre, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, 555 Wilmslow Road, Manchester, M20 4GJ, UK. (2021)
      DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as-if mis- or unrepaired-they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells. Defects in NHEJ factors are also associated with microcephaly, primordial dwarfism and immune deficiencies. One of the key proteins important for mediating NHEJ is XRCC4. XRCC4 is a dimer, with the dimer interface mediated by an extended coiled-coil. The N-terminal head domain forms a mixed alpha-beta globular structure. Numerous factors interact with the C-terminus of the coiled-coil domain, which is also associated with significant self-association between XRCC4 dimers. A range of construct lengths of human XRCC4 were expressed and purified, and the 1-164 variant had the best NMR properties, as judged by consistent linewidths, and chemical shift dispersion. In this work we report the 1H, 15 N and 13C backbone resonance assignments of human XRCC4 in the solution form of the 1-164 construct. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 156 of 161 assignable residues of XRCC4 were assigned to resonances in the TROSY spectrum, with an additional 11 resonances assigned to His-Tag residues. Prediction of solution secondary structure from a chemical shift analysis using the TALOS + webserver is in good agreement with the published X-ray crystal structures of this protein.
    • 1,2-Dimethylhydrazine-induced colon carcinoma and lymphoma in msh2(-/-) mice.

      Colussi, Claudia; Fiumicino, Silvia; Giuliani, Alessandro; Rosini, Sandra; Musiani, Piero; Macrí, Caterina; Potten, Christopher S; Crescenzi, Marco; Bignami, Margherita; Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanitá, Rome, Italy. (2001-10-17)
      BACKGROUND: Defective mismatch repair (MMR) in humans is particularly associated with familial colorectal cancer, but defective repair in mice is generally associated with lymphoma in the absence of experimental exposure to carcinogens. Loss of MMR also confers resistance to the toxic effects of methylating agents. We investigated whether resistance to methylation contributes to increased susceptibility to colorectal cancer in mice by exposing mice with defects in the MMR gene msh2 to a methylating agent. METHODS: Tumor incidence and time of death in msh2(+/+), msh2(+/-), and msh2(-/-) mice were analyzed after weekly exposure (until tumor appearance) to the methylating agent 1,2-dimethylhydrazine (DMH). Chemically induced and spontaneous tumors were characterized by frequency, type, and location. The tumor incidence in untreated and treated mice of each genotype was compared by a Mann-Whitney U test. Carcinogen-induced apoptosis in histologic sections of small and large intestines was also determined. All statistical tests were two-sided. RESULTS: Homozygous inactivation of the msh2 gene statistically significantly accelerated (P<.0001) death due to the development of DMH-induced colorectal tumors and lymphomas. Rates of death from DMH-induced colorectal adenocarcinoma were similar in msh2 heterozygous and wild-type mice, but only msh2 heterozygotes (msh(+/-)) developed additional, noncolorectal malignancies (notably trichofolliculoma [two of 21], angiosarcoma of the kidney capsule [two of 21], and lymphoma [one of 21]), suggesting that heterozygosity for msh2 slightly increases DMH susceptibility. DMH induced apoptosis in small intestinal and colonic epithelial crypts that was dependent on active msh2. CONCLUSIONS: Inactivation of msh2 allows the proliferation of gastrointestinal tract cells damaged by methylating agents. Furthermore, MMR constitutes a powerful defense against colorectal cancer induced by DNA methylation.
    • 1,N(2)-ethenoguanine, a mutagenic DNA adduct, is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase.

      Saparbaev, Murat K; Langouët, Sophie; Privezentzev, Cyril V; Guengerich, F Peter; Cai, Hongliang; Elder, Rhoderick H; Laval, Jacques; Cancer Research United Kingdom Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. M20 4BX (2002-07-26)
      The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)). Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N(2)-epsilonG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N(2)-epsilonG residues in vivo.
    • A [17F]-fluoromethane PET/TMS study of effective connectivity.

      Ferrarelli, Fabio; Haraldsson, H Magnus; Barnhart, Todd E; Roberts, Andy D; Oakes, Terrence R; Massimini, Marcello; Stone, Charles K; Kalin, Ned H; Tononi, Giulio; Department of Psychiatry, University of Wisconsin, Madison, 6001 Research Park Blvd., Madison, WI 53719, USA. (2004-08-30)
      We used transcranial magnetic stimulation (TMS) in combination with positron emission tomography (PET) to investigate the effective connectivity of four cortical regions within the same study. By employing [17F]-[CH3F] ([17F]-fluoromethane) as a radiotracer of blood-flow, we were able to obtain increased sensitivity compared to [15O]-H2O for both cortical and subcortical structures. The brain areas investigated were left primary motor cortex, right primary visual cortex, and left and right prefrontal areas. We found that each site of stimulation yielded a different pattern of activation/deactivation consistent with its anatomical connectivity. Moreover, we found that TMS of prefrontal and motor cortical areas gave rise to trans-synaptic activation of subcortical circuits.
    • [18F]-FLT positron emission tomography can be used to image the response of sensitive tumors to PI3-kinase inhibition with the novel agent GDC-0941.

      Cawthorne, C; Burrows, N; Gieling, R; Morrow, Christopher J; Forster, D; Gregory, J; Radigois, M; Smigova, A; Babur, M; Simpson, Kathryn L; et al. (2013)
      The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [(18)F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [(18)F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [(18)F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [(18)F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [(18)F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action.
    • 1p13 is the most frequently involved band in structural chromosomal rearrangements in human breast cancer.

      Mitchell, Erika L D; Santibanez-Koref, Mauro F; Cancer Research Campaign Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1990-11)
      Cytogenetic data on 14 breast carcinomas were examined to determine which chromosome arms and bands are preferentially involved in structural chromosome changes. Chromosome arms 17p, 16q, and 1p and band 1p13 were found to be significantly involved. A review of the world literature confirmed 1p as being the most frequently involved arm in structural chromosome changes in breast cancer and 1p13 as being the band most frequently involved in such changes. The two sets of results were pooled, and the analysis of 113 tumours revealed 229 of 304 bands to be involved, with 1p13 affected in 20% of the tumours.
    • 2'-Deacetoxyaustrospicatine from the stem bark of Taxus baccata.

      Breeden, S W; Jordan, Allan M; Lawrence, N J; McGown, Alan T; Department of Chemistry, UMIST, P.O. Box 88, Manchester, UK. (1996-02)
    • A [2+2] photo-adduct of 8-methoxypsoralen and thymine - x-ray crystal-structure - a model for the reaction of psoralens with dna in the phototherapy of psoriasis

      Land, Edward J; Rushton, Francis A P; Beddoes, R L; Bruce, J M; Cernik, R J; Dawson, S C; Mills, O S; Paterson Laboratories, Christie Hospital & Holt Radium Institute, Manchester, M20 9BX, U.K. (1982)
    • 20 alpha-hydroxysteroid dehydrogenase expression in hemopoietic cell cultures and its relationship to interleukin 3.

      Garland, John M; Dexter, T Michael; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982-12)
      Expression of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) has been investigated in long-term bone marrow cultures derived from normal and nude mice in the presence of horse serum and hydrocortisone. 20 alpha-SDH expression rises markedly in the adherent cells derived from normal bone marrow. Early (1-5 days) lipid profiles are similar to those seen in T cells, but at later times (6-14 days) stromal-type lipid patterns dominate. Similar culture of fetal liver and nu/nu bone marrow cells shows rises in 20 alpha-SDH expression, in situations where T cells are notably absent. The expression of 20 alpha-SDH is not associated with detectable endogenous production of "interleukin 3" activity, and although addition of "IL 3" to bone marrow cultures increases the levels of 20 alpha-SDH, this is associated with granulopoiesis rather than lymphoid development.
    • 21st L H Gray Conference: the radiobiology/radiation protection interface.

      West, Catharine M L; Martin, C J; Sutton, D G; Wright, Eric G; Academic Department of Radiation Oncology, University of Manchester, Christie Hospital, UK. catharine.west@manchester.ac.uk (2009-05)
      The 21st L H Gray Conference, organised by the L H Gray Trust with the Society for Radiological Protection, brought together international experts in radiobiology, epidemiology and risk assessment, and scientists involved in diagnostic and therapeutic radiation exposure. The meeting - held in Edinburgh, Scotland, on 4-6 June 2008 - aimed to raise awareness, educate and share knowledge of important issues in radiation protection. A distinguished group of speakers discussed topics that included (i) non-targeted effects of radiation, (ii) exposure to high natural background radiation, (iii) non-cancer effects in Japanese bomb survivors, (iv) lessons learnt from Chernobyl, (v) radiation in the workplace, (vi) biokinetic modelling, (vii) uncertainties in risk estimation, (viii) issues in diagnostic medical exposures, (ix) lessons leant from the polonium-210 incidence and (x) how the radiobiology/radiation oncology community is needed to help society prepare for potential future acts of radiation terrorism. The conference highlighted the importance, relevance and topicality of radiobiology today.
    • 265 NM Laser flash photolysis of some ortho-substituted anilides and related N-formylkynurenine derivatives.

      Pileni, M P; Santus, R; Land, Edward J; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1978)
    • 3'-UTR poly(T/U) tract deletions and altered expression of EWSR1 are a hallmark of mismatch repair-deficient cancers.

      Kishore, S; Piscuoglio, S; Kovac, M; Gylling, A; Wenzel, F; Trapani, Francesca; Altermatt, H J; Mele, V; Marra, G; Peltomäki, P; et al. (2014-01-01)
      The genome-wide accumulation of DNA replication errors known as microsatellite instability (MSI) is the hallmark lesion of DNA mismatch repair (MMR)-deficient cancers. Although testing for MSI is widely used to guide clinical management, the contribution of MSI at distinct genic loci to the phenotype remains largely unexplored. Here, we report that a mononucleotide (T/U)16 tract located in the 3' untranslated region (3'-UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene is a novel MSI target locus that shows perfect sensitivity and specificity in detecting mismatch repair-deficient cancers in two independent populations. We further found a striking relocalization of the EWSR1 protein from nucleus to cytoplasm in MMR-deficient cancers and that the nonprotein-coding MSI target locus itself has a modulatory effect on EWSR1 gene expression through alternative 3' end processing of the EWSR1 gene. Our results point to a MSI target gene-specific effect in MMR-deficient cancers.
    • 3-substituted-5-aziridinyl-1-methylindole-4,7-diones as NQO1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro.

      Jaffar, Mohammed; Phillips, Roger M; Williams, Kaye J; Mrema, Ibrahim; Cole, Christian; Wind, Natasha S; Ward, Timothy H; Stratford, Ian J; Patterson, Adam V; School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK. (2003-10-01)
      Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure-activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug.
    • 32P-post-labelling analysis of DNA adducts formed in the upper gastrointestinal tissue of mice fed bracken extract or bracken spores.

      Povey, Andrew C; Potter, D; O'Connor, Peter J; Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK. (1996-11)
      Bracken toxicity to both domestic and laboratory animals is well established and tumours are formed when rodents are treated with either bracken extracts or bracken spores. In this study we have administered bracken spores and extract to mice in order to investigate whether such exposure leads to the formation of DNA adducts. DNA, isolated from the upper gastrointestinal tract and liver, was digested to 3'-nucleotides. Adducts were extracted with butanol, 32P-post-labelled, separated by thin layer chromatography (TLC) and visualised and quantified using storage-phosphor technology. A cluster of adducts was clearly seen in the DNA of the upper gastrointestinal tract, but not liver, 5 and 24 h after treatment with bracken extract or bracken spores. These adducts were not observed in DNA extracted from vehicle-treated animals. Whereas, after 5 h adduct levels in extract and spore-treated animals were similar, after 24 h adduct levels in the extract-treated animals had diminished by > 75%, but levels in spore-treated animals remained similar to those found after 5 h. This suggests that the DNA-reactive compounds were being released slowly from the spores, even though the spores had been sonicated before administration. Adducts were also quantified after the addition of an internal standard (deoxyinosine 3'-monophosphate) by comparing the amount of label incorporated into the adducts with that found in a known amount of the internal standard. Adduct levels using this internal standard approach were similar to those found by direct measurement of radioactivity incorporated into the adduct, indicating that the labelling of adducts was quantitative. We have tried, unsuccessfully, to synthesise ptaquiloside, the principal carcinogenic component present within bracken. However, similar patterns of adducts were observed when two other compounds, (1-(4-chlorophenyl sulphonyl)-l-cyclopropane carbonitrile and 3-cyclopropylindeno [1,2-c] pyrazol-4-(O-methyl)oxime), which both contain a cyclopropyl ring, were administered to mice. The adducts detected in bracken-treated animals may, thus, have arisen from ptaquiloside but, whether these adducts arise directly from the compounds and bracken spores/extract themselves or via an indirect mechanism, remains to be determined. As bracken-induced DNA adducts are detectable in rodent tissues by a 32P-post-labelling procedure commonly employed to investigate DNA damage in human populations, it may prove possible to apply such approaches to determine human exposure.
    • 32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase.

      Povey, Andrew C; Cooper, Donald P; Littler, Edward; Cancer Research Campaign Department of Carcinogenesis, Patterson Institute for Cancer Research, Manchester, UK. (1991-04)
      Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.
    • 3D Bioprinting: an enabling technology to understand melanoma

      Fernandes, S.; Vyas, C.; Lim, P.; Pereira, R. F.; Virós, Amaya; Bártolo, P.; Department of Mechanical, Aerospace and Civil Engineering, University of Manchester, Oxford Road, Manchester M13 9PL, UK (2022)
      Melanoma is a potentially fatal cancer with rising incidence over the last 50 years, associated with enhanced sun exposure and ultraviolet radiation. Its incidence is highest in people of European descent and the ageing population. There are multiple clinical and epidemiological variables affecting melanoma incidence and mortality, such as sex, ethnicity, UV exposure, anatomic site, and age. Although survival has improved in recent years due to advances in targeted and immunotherapies, new understanding of melanoma biology and disease progression is vital to improving clinical outcomes. Efforts to develop three-dimensional human skin equivalent models using biofabrication techniques, such as bioprinting, promise to deliver a better understanding of the complexity of melanoma and associated risk factors. These 3D skin models can be used as a platform for patient specific models and testing therapeutics.
    • 3D modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

      Maguire, S; Peck, B; Wai, Patty T; Campbell, J; Barker, H; Gulati, A; Daley, F; Vyse, S; Huang, P; Lord, C; et al. (2016-08-11)
      The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations, changes in gene expression, alongside microenvironmental and recognised histological alterations. Here we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression and to functionally validate potential driver alterations in 3-dimensional (3D) spheroids that may give insight into breast cancer progression and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number, mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired from non-malignant MCF10A cells to their malignant counterparts that are also present in primary breast cancers re-analysed from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously un-described RAB3GAP1-HRAS and UBA2-PDCD2L expressed in-frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression identified that when cells are grown as 3D spheroids, they show perturbations of cancer-relevant pathways. Functional interrogation of the dependency on predicted driver events, identified alterations in HRAS, PIK3CA, and TP53 that selectively decreased cell growth and were associated with progression from pre-invasive to invasive disease, only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like in vivo. The MCF10 progression series, therefore, represents a good model to dissect potential biomarkers and evaluation of therapeutic targets involved in the progression of breast cancer.
    • 3H-thymidine labelling index (TLI) as a marker of tumour growth heterogeneity: evaluation in human solid carcinomas.

      Becciolini, A; Balzi, M; Barbarisi, M; Faraoni, P; Biggeri, A; Potten, Christopher S; Department of Clinical Physiopathology, University of Florence, Italy. (1997)
      Many studies deal with the analysis of cell kinetic, cytogenetic, biochemical and molecular cell biology parameters to identify prognostic factors relating to tumour growth but all methods use only a small part of the total tumour mass. This study is devoted to the analysis of the heterogeneity of the growth of human solid tumours assaying proliferative activity by means of 3H-thymidine labelling index (TLI) in a fixed number of samples collected in different areas of the lesion (larynx and colon cancers), or in different lesions of the same subject (breast and bladder cancers). Each sample (at the macroscopic level) was divided into small fragments (at the microscopic level) and proliferative activity was determined. The analysis of variance for hierarchical designs demonstrated that in all cases a high component of the variance is attributable to the subjects and to the fragments whereas the variance attributable to the different areas is very low. The heterogeneity of proliferative activity displays a higher focal variability among the fragments (microscopic level) compared with that among areas (macroscopic level) within subjects, provided an adequate number of fragments and cells are counted. In multiple synchronous carcinoma of the bladder the wide variability of proliferation among the single lesions demonstrated that it is necessary to analyse all the tumours in a subject because each one is characterized by a different cell growth potential.