• Intratumoral FoxP3(+)Helios(+) regulatory T cells upregulating immunosuppressive molecules are expanded in human colorectal cancer.

      Syed Khaja, A; Toor, S; El Salhat, H; Ali, B; Elkord, Eyad; Cancer Research Center, College of Science and Engineering, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar (2017)
      Regulatory T cells (Tregs) can be antitumorigenic or pro-tumorigenic in colorectal cancer (CRC) depending on the presence of different Treg subsets with various immunosuppressive molecules. Some studies reported the phenotypic characteristics of tumor-infiltrating immune cells in CRC, but limited studies have focused on the co-expression of suppressive molecules on immune cells. The aim of this study was to characterize immune cells in the tumor microenvironment (TME), compared to paired adjacent non-tumor colon tissue of CRC patients. Additionally, we investigated co-expression of immunosuppressive molecules on different Treg subsets in the TME, normal colon tissue, and peripheral blood of CRC patients and healthy donors. In this preliminary study, we report that the majority of CD3(+) T cells in the TME are CD4(+) T cells with high co-expression of programmed death 1 (PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1/CD39 molecules. Levels of CD4(+)FoxP3(+)Helios(+) Tregs were significantly increased in the TME. Furthermore, we observed increased levels of PD-1/CTLA-4 and PD-1/CD39 co-expressing cells within FoxP3(+)Helios(+) and FoxP3(+)Helios(-) Treg subsets, indicative of their potent immunosuppressive potential. These results suggest synergistic associations between PD-1/CTLA-4 and PD-1/CD39 in dampening T-cell activation and function along with suppressing tumor-specific immune responses, suggesting that dual blockade of these molecules could be a more effective strategy for inducing antitumor immune responses in CRC.
    • Intratumoral heterogeneity of glioblastoma infiltration revealed by joint histogram analysis of diffusion tensor imaging.

      Li, C; Wang, S; Yan, J; Piper, R; Liu, H; Torheim, Turid; Kim, H; Zou, J; Boonzaier, N; Sinha, R; et al. (2018-09-17)
      Glioblastoma is a heterogeneous disease characterized by its infiltrative growth, rendering complete resection impossible. Diffusion tensor imaging (DTI) shows potential in detecting tumor infiltration by reflecting microstructure disruption.
    • Intravenous administration of the selective toll-like receptor 7 agonist DSR-29133 leads to anti-tumor efficacy in murine solid tumor models which can be potentiated by combination with fractionated radiotherapy.

      Dovedi, Simon J; Adlard, A; Ota, Y; Murata, M; Sugaru, E; Koga-Yamakawa, E; Eguchi, K; Hirose, Y; Yamamoto, S; Umehara, H; et al. (2016-03-05)
      Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.
    • Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix.

      West, Catharine M L; Davidson, Susan E; Roberts, Stephen A; Hunter, Robin D; Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital (NHS Trust), Manchester, UK. (1993-10)
      The intrinsic radiosensitivity of cervical carcinoma has been measured using a soft agar clonogenic assay. All patients received radical radiotherapy alone with a minimum of 2 years post-treatment follow-up. Only women with stage I, II and III disease were included in the analysis. Values for cell surviving fraction at 2 Gy (SF2) were obtained for 88 tumours with an assay success rate of 73%. The 53 patients alive and well at the time of analysis had tumours with a mean SF2 that was significantly lower than the value from the 22 patients with locoregional failure (P < 0.01). Patients with radioresistant tumours (SF2 > 0.40, the median) had a significantly lower 3 year survival level than those with sensitive tumours (SF2 < or = 0.40) (P = 0.002). Also the frequency of local recurrence was higher (P = 0.001) whether these were central (P = 0.009) or peripheral (P = 0.046). Cell surviving fraction at 3.5 Gy was obtained for 46 tumours and the 3 year patient survival rate was significantly higher for those with SF3.5 values less than the median (P = 0.043). There was, however, no difference in the level of local recurrence (P = 0.24). The ability to grow in culture was not associated with significantly poorer patient survival (P = 0.56) or failure to control the primary disease (P = 0.17). While high colony forming efficiencies were associated with an increased rate of local recurrence (P = 0.029) they did not predict for overall patient survival (P = 0.32). These data suggest that, for cervical carcinoma treated with radical radiotherapy, intrinsic radiosensitivity is important in determining treatment outcome.
    • The intrinsic radiosensitivity of cervical carcinoma: correlations with clinical data.

      West, Catharine M L; Davidson, Susan E; Burt, Paul A; Hunter, Robin D; Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Manchester. (1995-02-15)
      PURPOSE: The aims of the work were to study the intrinsic radiosensitivity of tumor biopsies from patients with cervical carcinoma and to correlate the data with information on patient age, disease stage, differentiation status, tumor volume, and tumor ploidy. METHODS AND MATERIALS: Radiosensitivity was assessed for 145 tumors in vitro as surviving fraction at 2 Gy (SF2) using a clonogenic assay. RESULTS: Although the clonogens in tumors classified as Stage I or II tended to be more radiosensitive than in Stage III or IV disease, the difference was not statistically significant (p > 0.15). There was also no significant difference in the intrinsic radiosensitivity of well, moderately, or poorly differentiated tumors or between squamous cell carcinoma and adenocarcinoma (p > 0.53). There was no correlation between patient age and tumor radiosensitivity (p = 0.49). Large volume (> or = 4 cm) disease was more radioresistant than small volume (< 4 cm) disease, but the difference was not significant (p = 0.08). Finally, diploid tumors tended to be more radioresistant than aneuploid tumors (p = 0.07). CONCLUSION: The intrinsic radiosensitivity of cervix tumors is independent of disease stage, tumor grade, and patient age. Weak trends, however, were observed of increased tumor radioresistance for large volume disease and diploid tumors, suggesting that tumor SF2 may not be a completely independent parameter.
    • The intrinsic radiosensitivity of normal and tumour cells.

      West, Catharine M L; Davidson, Susan E; Elyan, S A; Swindell, Ric; Roberts, Stephen A; Orton, C J; Coyle, C A; Valentine, Helen R; Wilks, Deepti P; Hunter, Robin D; et al. (1998-04)
      PURPOSE: To examine whether in vitro measurements of normal and tumour cell radiosensitivity can be used as prognostic factors in clinical oncology. MATERIALS AND METHODS: Stage I-III cervix carcinoma patients were treated with radical radiotherapy with a minimum of 3 years' follow-up. Lymphocyte and tumour radiosensitivities were assayed using, respectively, a limiting dilution and soft agar clonogenic assay to obtain surviving fraction at 2 Gy (SF2). The results were related, in an actuarial analysis, to late morbidity assessed using the Franco Italian glossary. RESULTS: Patients with radiosensitive lymphocytes had a significantly increased risk of developing late complications (n = 93, p = 0.002). Increasing tumour radiosensitivity was associated with an increased risk of morbidity (n= 113, p=0.032). A significant correlation was found between fibroblast and tumour cell radiosensitivity (r=0.57, p=0.03), but a weak inverse association was found between lymphocyte and tumour cell radiosensitivity (r= -0.32, p=0.03). Patients with radiosensitive lymphocytes and tumour cells had higher levels of late complications than those whose cells were radioresistant. CONCLUSION: The work described highlights the importance of cellular radiosensitivity as a parameter determining the clinical response to radiotherapy.
    • Intrinsically connected: therapeutically targeting the cathepsin proteases and the Bcl-2 family of protein substrates as co-regulators of apoptosis

      Soond, S. M.; Kozhevnikova, M. V.; Savvateeva, L. V.; Townsend, Paul A; Zamyatnin, A. A., Jr.; Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Trubetskaya str. 8-2, 119991 Moscow, Russi (2021)
      Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting 'BH3-mimetics' can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.
    • Introduction of the activated N-ras oncogene into human fibroblasts by retroviral vector induces morphological transformation and tumorigenicity.

      Kinsella, Anne R; Fiszer-Maliszewska, Lucja; Mitchell, Erika L D; Guo, Ya-Ping; Fox, Margaret; Scott, David; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1990-10)
      The introduction of activated N-ras cDNA into normal diploid human skin fibroblast cell cultures using the retroviral vector pZIPneo results in a spectrum of morphologies ranging from near normal to, in rare instances, dense piled-up colonies of morphologically transformed cells. However, none of the clones isolated were transformed as assessed by growth on agar or tumorigenicity in nude mice. Introduction of both c-myc and N-ras oncogene cDNAs into normal skin fibroblasts failed to produce transformation as assessed by growth on agar and tumorigenicity in nude mice, although c-myc infection alone conferred immortality and the resultant doubly infected cell line was immortal. Using the same construct, activated N-ras cDNA was shown to transform immortalized human fibroblasts to tumorigenicity. However, immortalization per se was shown not to guarantee 'co-operation' with an activated N-ras gene to give malignant transformation. Although numerical and structural chromosome aberrations (clonal and non-clonal) were observed in some of the cell strains isolated after retroviral infection, these were not directly associated with viral infection, the presence of the oncogenes or with the morphologically transformed phenotype.
    • An introduction to artificial neural networks in bioinformatics--application to complex microarray and mass spectrometry datasets in cancer studies.

      Lancashire, Lee J; Lemetre, Christophe; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. llancashire@picr.man.ac.uk (2009-05)
      Applications of genomic and proteomic technologies have seen a major increase, resulting in an explosion in the amount of highly dimensional and complex data being generated. Subsequently this has increased the effort by the bioinformatics community to develop novel computational approaches that allow for meaningful information to be extracted. This information must be of biological relevance and thus correlate to disease phenotypes of interest. Artificial neural networks are a form of machine learning from the field of artificial intelligence with proven pattern recognition capabilities and have been utilized in many areas of bioinformatics. This is due to their ability to cope with highly dimensional complex datasets such as those developed by protein mass spectrometry and DNA microarray experiments. As such, neural networks have been applied to problems such as disease classification and identification of biomarkers. This review introduces and describes the concepts related to neural networks, the advantages and caveats to their use, examples of their applications in mass spectrometry and microarray research (with a particular focus on cancer studies), and illustrations from recent literature showing where neural networks have performed well in comparison to other machine learning methods. This should form the necessary background knowledge and information enabling researchers with an interest in these methodologies, but not necessarily from a machine learning background, to apply the concepts to their own datasets, thus maximizing the information gain from these complex biological systems.
    • Introduction to the haemopoietic system.

      Dexter, T Michael; Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester. (1990)
    • Introduction to the National Cancer Imaging Translational Accelerator (NCITA): a UK-wide infrastructure for multicentre clinical translation of cancer imaging biomarkers

      McAteer, M. A.; O'Connor, James P B; Koh, D. M.; Leung, H. Y.; Doran, S. J.; Jauregui-Osoro, M.; Muirhead, N.; Brew-Graves, C.; Plummer, E. R.; Sala, E.; et al. (2021)
      The National Cancer Imaging Translational Accelerator (NCITA) is creating a UK national coordinated infrastructure for accelerated translation of imaging biomarkers for clinical use. Through the development of standardised protocols, data integration tools and ongoing training programmes, NCITA provides a unique scalable infrastructure for imaging biomarker qualification using multicentre clinical studies.
    • Invasive characteristics of human prostatic epithelial cells: understanding the metastatic process.

      Hart, Claire A; Brown, Michael D; Bagley, Steven; Sharrard, M; Clarke, Noel W; PromPT Genito-Urinary Cancer Research, Cancer Research UK Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. (2005-02-14)
      Prostate cancer has a predilection to metastasise to the bone marrow stroma (BMS) by an as yet uncharacterised mechanism. We have defined a series of coculture models of invasion, which simulate the blood/BMS boundary and allow the elucidation of the signalling and mechanics of trans-endothelial migration within the complex bone marrow environment. Confocal microscopy shows that prostate epithelial cells bind specifically to bone marrow endothelial-to-endothelial cell junctions and initiate endothelial cell retraction. Trans-endothelial migration proceeds via an epithelial cell pseudopodial process, with complete epithelial migration occurring after 232+/-43 min. Stromal-derived factor-1 (SDF-1)/CXCR4 signalling induced PC-3 to invade across a basement membrane although the level of invasion was 3.5-fold less than invasion towards BMS (P=0.0007) or bone marrow endothelial cells (P=0.004). Maximal SDF-1 signalling of invasion was completely inhibited by 10 microM of the SDF-1 inhibitor T140. However, 10 microM T140 only reduced invasion towards BMS and bone marrow endothelial cells by 59% (P=0.001) and 29% (P=0.011), respectively. This study highlights the need to examine the potential roles of signalling molecules and/or inhibitors, not just in single-cell models but in coculture models that mimic the complex environment of the bone marrow.
    • Invasive phenotype of primary human prostatic epithelial cells.

      Scott, L J; Clarke, Noel W; Sharrard, M S; George, N J; Lang, S H; Department of Urology and Paterson Institute CRC Research Laboratories, Christie Hospital, Manchester, UK. (2000-12)
    • Investigating FTIR based histopathology for the diagnosis of prostate cancer.

      Baker, Matthew J; Gazi, Ehsan; Brown, Michael D; Shanks, Jonathan H; Clarke, Noel W; Gardner, Peter; Manchester Interdisciplinary Biocentre, Centre for Instrumentation and Analytical Science, School of Chemical Engineering and Analytical Science, The University of Manchester, 131 Princess Street, Manchester, UK. (2009-02)
      Prostate cancer is the most common gender specific cancer. The current gold standard for diagnosis, histopathology, is subjective and limited by variation between different pathologists. The diagnostic problems associated with the correct grading and staging of prostate cancer (CaP) has led to an interest in the development of spectroscopic based diagnostic techniques. FTIR microspectroscopy used in combination with a Principal Component Discriminant Function Analysis (PC-DFA) was applied to investigate FTIR based histopathology for the diagnosis of CaP. In this paper we report the results of a large patient study in which FTIR has been proven to grade CaP tissue specimens to a high degree of sensitivity and specificity.
    • Investigating the importance of B cells and antibodies during Trichuris muris infection using the IgMi mouse

      Sahputra, R.; Murphy, E. A.; Forman, R.; Mair, I.; Fadlullah, Muhammad Z H; Waisman, A.; Muller, W.; Else, K. J.; Division of Infection, Immunity and Respiratory Medicine, Lydia Becker Institute for Immunology, The University of Manchester, Manchester, UK. (2020)
      The IgMi mouse has normal B cell development; its B cells express an IgM B cell receptor but cannot class switch or secrete antibody. Thus, the IgMi mouse offers a model system by which to dissect out antibody-dependent and antibody-independent B cell function. Here, we provide the first detailed characterisation of the IgMi mouse post-Trichuris muris (T. muris) infection, describing expulsion phenotype, cytokine production, gut pathology and changes in T regulatory cells, T follicular helper cells and germinal centre B cells, in addition to RNA sequencing (RNA seq) analyses of wild-type littermates (WT) and mutant B cells prior to and post infection. IgMi mice were susceptible to a high-dose infection, with reduced Th2 cytokines and elevated B cell-derived IL-10 in mesenteric lymph nodes (MLN) compared to controls. A low-dose infection regime revealed IgMi mice to have significantly more apoptotic cells in the gut compared to WT mice, but no change in intestinal inflammation. IL-10 levels were again elevated. Collectively, this study showcases the potential of the IgMi mouse as a tool for understanding B cell biology and suggests that the B cell plays both antibody-dependent and antibody-independent roles post high- and low-dose T. muris infection. KEY MESSAGES: During a high-dose T. muris infection, B cells are important in maintaining the Th1/Th2 balance in the MLN through an antibody-independent mechanism. High levels of IL-10 in the MLN early post-infection, and the presence of IL-10-producing B cells, correlates with susceptibility to T. muris infection. B cells maintain gut homeostasis during chronic T. muris infection via an antibody-dependent mechanism. Keywords: B cells; IgMi mouse; Interleukin-10; Intestinal pathology; Th1/Th2; Trichuris muris.
    • Investigation of a possible role for superoxide anion production in tumour promotion.

      Kinsella, Anne R; Gainer, Helen St C; Butler, John; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester UK (1983)
      The effect of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on superoxide (O-.2) production and superoxide dismutase (SOD) levels has been investigated in human leukocytes and human and rodent fibroblast cell lines. Both O-.2 production and a lowering of the SOD levels were observed in the human leukocytes, but these changes could not be correlated with the induction of gross chromosomal aberrations by TPA. Similarly the use of an O-.2-generating system failed to induce chromosome aberrations. However, a small percentage increase in chromosome aberrations was observed in human fibroblasts after repeated and prolonged exposure to TPA. TPA was shown to reduce the SOD levels in these cells. Overall however, the data suggest that the chromosomes are not the primary site of action for the superoxide anions produced by TPA.
    • Investigation of human chromosome polymorphisms by scanning electron microscopy.

      Harrison, Christine J; Jack, E; Allen, Terence D; Harris, R; The Department of Cell Biology and Cytogenetics, and the Department of Ultrastructure, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1985-02)
      Human chromosome polymorphisms were investigated by scanning electron microscopy (SEM). Centromeric heterochromatin was of a constricted morphology. The extent of the C banded region was demarcated by a prominent circumferential groove in G banded chromosomes. Circumferential grooves were observed within the heterochromatin of chromosome 9, and the number of grooves present reflected the size of the region. Three dimensional viewing of satellites and short arms of acrocentric chromosomes, from different angles in the SEM, provided the opportunity for accurate assessment of the size of satellites to be made. Also, small morphological variations were defined in the SEM when definition was uncertain in the light microscope (LM).
    • Investigation of mammary epithelial cell-bone marrow stroma interactions using primary human cell culture as a model of metastasis.

      Brooks, B; Bundred, Nigel J; Howell, Anthony; Lang, Shona H; Testa, Nydia G; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. ptbmb@liv.ac.uk (1997-11-27)
      A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow. Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes. Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue. The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture. Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts. In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence. Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion.
    • Investigation of the cell cycle response of normal and Fanconi's anaemia fibroblasts to nitrogen mustard using flow cytometry.

      Dean, S W; Fox, Margaret; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1983-11)
      Cell survival has been measured in normal and Fanconi's anaemia (FA) human fibroblasts after treatment with the bifunctional alkylating agent, nitrogen mustard (HN2). Two FA cell lines exhibited 6- to 10-fold greater sensitivity than the normal cell line. Flow cytometry was used to investigate the effects of HN2 on cell cycle progression of normal and FA cells. After 0.1 microgram/ml HN2 (surviving fraction, s.f. = 0.8) normal cells exhibited an S phase accumulation within 6 h, followed by a transient G2 delay. At higher doses of HN2, the S phase delay became more pronounced and there was considerably greater accumulation of cells in G2. HN2 at 0.01 microgram/ml (s.f. = 0.8) induced no detectable S or G2 delay in FA cells. A higher dose, 0.1 microgram/ml (s.f. = 0.13 and 0.29), again induced no S phase delay, but a gradual accumulation of cells in G2 was observed up to 78 h after treatment. The presence of an S phase delay in normal cells after HN2 treatment may be important in allowing time for DNA repair before completion of DNA synthesis. The absence of such a delay in FA cells suggests that an inability to delay S phase traverse in response to DNA damage from bifunctional alkylating agents may contribute to the sensitivity of FA cells to such drugs.
    • Investigation of the effects of the phorbol ester TPA on carcinogen-induced forward mutagenesis to 6-thioguanine-resistance in V79 Chinese hamster cells.

      Kinsella, Anne R; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, UK (1981)
      12-O-Tetradecanoyl-phorbol-13-acetate (TPA) was tested for its effect on N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced forward mutagenesis to 6-thioguanine resistance (6TGR) in V79 Chinese hamster cells. Using a replating assay, no effect of TPA on the recovery of 6TGR mutants was observed. Previous reports of TPA enhancement of carcinogen-induced forward mutagenesis using the in situ mutagenesis assay, can be explained in terms of the influence of metabolic co-operation on mutant recovery. TPA is known to eliminate metabolic co-operation, an artefact of the in situ mutagenesis assay system, thus contributing to the enhancement of mutant recovery in TPA treated cultures. This investigation aims to demonstrate that TPA has no influence on mutagenesis per se, positive or negative enhancement of forward mutagenesis being solely a reflection of the mutagenesis assay employed.