• 5T4 as a target for immunotherapy in renal cell carcinoma.

      Elkord, Eyad; Shablak, Alaaeldin; Stern, Peter L; Hawkins, Robert E (2009-12)
    • 5T4 glycoprotein regulates the sensory input-dependent development of a specific subtype of newborn interneurons in the mouse olfactory bulb.

      Yoshihara, S; Takahashi, H; Nishimura, N; Naritsuka, H; Shirao, T; Hirai, H; Yoshihara, Y; Mori, K; Stern, Peter L; Tsuboi, A; et al. (2012-02-08)
      Sensory input has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor-evoked activity dependent. However, little is known about the developmental role of sensory input in the other major OB population of the GABAgenic interneurons, such as granule cells and periglomerular cells. Here, we identified, with DNA microarray and in situ hybridization screenings, a trophoblast glycoprotein gene, 5T4, whose expression in a specific subtype of OB interneurons is dependent on sensory input. 5T4 is a type I membrane protein, whose extracellular domain contains seven leucine-rich repeats (LRR) flanked by characteristic LRR-N-flanking and C-flanking regions, and a cytoplasmic domain. 5T4 overexpression in the newborn OB interneurons facilitated their dendritic arborization even under the sensory input-deprived condition. By contrast, both 5T4 knockdown with RNAi and 5T4 knockout with mice resulted in a significant reduction in the dendritic arborization of 5T4(+) granule cells. Further, we identified the amino acid sequence in the 5T4 cytoplasmic domain that is necessary and sufficient for the sensory input-dependent dendritic shaping of specific neuronal subtypes in the OB. Thus, these results demonstrate that 5T4 glycoprotein contributes in the regulation of activity-dependent dendritic development of interneurons and the formation of functional neural circuitry in the OB.
    • 5T4 interacts with TIP-2/GIPC, a PDZ protein, with implications for metastasis.

      Awan, Abida; Lucic, Melinda R; Shaw, David M; Sheppard, Freda C; Westwater, Caroline; Lyons, Steve; Stern, Peter L; CRC Immunology Group, CRC Molecular Biology Group, The Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, United Kingdom. (2002-01-25)
      Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.
    • 5T4 oncofetal antigen expression in ovarian carcinoma.

      Wrigley, E; McGown, Alan T; Rennison, J; Swindell, Ric; Crowther, Derek; Starzynska, T; Stern, Peter L; CRC Department of Medical Oncology, Christie Hospital, CRC Department of Experimental Chemotherapy, Paterson Institute, CRC Department of Medical Statistics, Christie Hospital, CRC Department of Immunology, Paterson Institute, Manchester, UK. (1995-07)
      5T4 oncofetal antigen is defined by a monoclonal antibody raised against human placental trophoblast, and recognizes a 72 kD glycoprotein expressed in many different carcinomas but detected only at low levels in some normal epithelia. Analysis of the patterns of expression of 5T4 oncofetal antigen in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumor cells and metastatic spread. The 5T4 antigen expression of 72 epithelial ovarian carcinomas has been investigated by immunohistochemistry; 71% of the carcinomas demonstrated positive 5T4 immunoreactivity in adenocarcinoma cells and/or associated stromal tissue. In order to assess any relationship to prognosis, the 5T4 phenotypes were analyzed with respect to various clinicopathologic features of the tumors and the clinical outcome of the patients assessed by survival and disease-free interval. There was a significant correlation between 5T4 expression and more advanced stage of disease (FIGO stages III and IV) (P < 0.001) and with poorly differentiated tumors (P = 0.036) compared to well or moderately differentiated tumors. Patients with tumors expressing 5T4 were less likely to respond well to adjuvant therapy (P = 0.030) and had a significantly worse outlook in terms of survival (P = 0.033) and disease-free interval (P = 0.033). This significance was not demonstrated as acting independently of FIGO stage and tumor differentiation.
    • 5T4 oncofetal antigen in gastric carcinoma and its clinical significance.

      Starzynska, T; Wiechowska-Kozlowska, A; Marlicz, K; Bromley, Michael; Roberts, Stephen A; Lawniczak, M; Kolodziej, B; Zyluk, A; Stern, Peter L; Department of Gastroenterology, Medical Pomeranian Academy, Szczecin, Poland. (1998-06)
      OBJECTIVE: To evaluate the role of 5T4 antigen in gastric cancer progression and prognosis. DESIGN: A prospective study of 5T4 antigen expression in primary, secondary and recurrent gastric carcinoma, the relationship to selected prognostic parameters and the course of disease. PATIENTS: Eighty six patients operated on for gastric cancer. TISSUE: One hundred and twenty two gastric tumours were studied, including 86 primary carcinomas, 32 coexisting lymph node metastases and four recurrent carcinomas. METHODS: Immunohistochemistry using 5T4 monoclonal antibody on frozen sections. RESULTS: The 5T4 antigen was detected in 41% of primary gastric tumours including early gastric cancer. A strong relationship was found between 5T4 positivity and tumour histology. Thus, 52% of gastric carcinomas of intestinal type expressed 5T4 antigen compared with 28% of the diffuse type (P = 0.028). Among 16 sets of primary gastric carcinomas and regional lymph node metastases, coordinate 5T4 expression was seen in 14 cases; the other two showed acquisition of positivity on metastatic tumour cells (carcinomas of diffuse type). 5T4 antigen was detected more frequently in carcinomas with p53 accumulation compared with those with undetectable p53 levels (P = 0.015). The presence of 5T4 in cancer cells was correlated with poor short-term prognosis (24% vs 49% of 2 year survival for 5T4 positive and negative tumours respectively, P = 0.024). The effect on survival was evident in the p53 negative group, with patients 5T4 positive showing worse survival (28% vs 60% in 2 years). CONCLUSIONS: Our results suggest that the assessment of 5T4 expression in gastric carcinoma can be helpful in identifying patients with poor short-term prognosis.
    • 5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype.

      Castro, Fernanda V; McGinn, Owen J; Krishnan, S; Marinov, Georgi; Li, J; Rutkowski, A J; Elkord, Eyad; Burt, Deborah J; Holland, M; Vaghjiani, R; et al. (2012-01-23)
      Although the overall prognosis in childhood acute lymphoblastic leukemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterized by the occurrence of disease at extramedullary sites, such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofetal antigen transcript levels in cytogenetic high-risk subgroups of patients (P<0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4-positive leukemic blasts than healthy donors (P=0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4(+) (Sup5T4) and 5T4(-) (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared with Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4-positive Sup-B15 cells are susceptible to 5T4-specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high-risk pre-B-ALL.Leukemia advance online publication, 17 February 2012; doi:10.1038/leu.2012.18.
    • The 5T4 oncofoetal antigen is an early differentiation marker of mouse ES cells and its absence is a useful means to assess pluripotency.

      Ward, Christopher M; Barrow, Katie M; Woods, Andrew M; Stern, Peter L; Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. cward@picr.man.ac.uk (2003-11-15)
      5T4 oncotrophoblast antigen is a transmembrane glycoprotein expressed by trophoblast and many carcinomas but not most normal adult tissues. Results from overexpression of human and mouse 5T4 cDNA in cell lines are consistent with it having an influence on adhesion, shape and motility. We show that murine embryonic stem cell lines are 5T4 negative but that there is rapid up regulation of protein and transcripts upon differentiation, including derivatives of each primary germ layer, as evidenced by cell surface FACS, western and RT-PCR analyses. The kinetics of differentiation and 5T4 expression are closely correlated, with early events linking 5T4 expression to changes in motility and morphology. Comparison of 5T4 expression with other ES cell transcript (Oct 3/4; Rex-1) and antigen markers (Forsmann, SSEA-1) establishes 5T4 as a useful marker for the non-destructive detection of early differentiation of ES cells. For example, 'undifferentiated' ES phenotype defined as SSEA-1 positive and 5T4 negative is seven times more efficient at chimera formation than SSEA-1-positive/5T4-positive cells. Thus, 5T4 glycoprotein expression is associated with early differentiative events of ES cells involving altered motility, and it has useful practical consequences for assessing ES potency and studying similar processes in development and metastasis.
    • 5T4 oncofoetal antigen: an attractive target for immune intervention in cancer.

      Stern, Peter L; Harrop, R; Institute of Cancer Studies, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX (2016-10-18)
      The natural history of a patient's cancer is often characterised by genetic diversity and sequential sweeps of clonal dominance. It is therefore not surprising that identifying the most appropriate tumour-associated antigen for targeted intervention is challenging. The 5T4 oncofoetal antigen was identified by searching for surface molecules shared between human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host. The 5T4 protein is expressed by many different cancers but rarely in normal adult tissues. 5T4 molecules are 72 kD, heavily N-glycosylated proteins with several leucine-rich repeats which are often associated with protein-protein interactions. 5T4 expression is associated with the directional movement of cells through epithelial mesenchymal transition, potentiation of CXCL12/CXCR4 chemotaxis and inhibition of canonical Wnt/beta-catenin while favouring non-canonical pathway signalling; all processes which help drive the spread of cancer cells. The selective pattern of 5T4 tumour expression, association with a tumour-initiating phenotype plus a mechanistic involvement with cancer spread have underwritten the clinical development of different immunotherapeutic strategies including a vaccine, a tumour-targeted superantigen and an antibody drug conjugate. In addition, a chimeric antigen receptor T cell approach targeting 5T4 expressing tumour cells is in pre-clinical development. A key challenge will include how best to combine each 5T4 targeted immunotherapy with the most appropriate standard of care treatment (or adjunct therapy) to maximise the recovery of immune control and ultimately eliminate the tumour.
    • A novel circulating tumour cell-derived xenograft (CDX) model to investigate uveal melanoma metastasis.

      Mahal, Katharina; Lee, Rebecca J; Garcia-Martinez, Pablo; Smith, Matthew; Dhomen, Nathalie; Marais, Richard; Univ Manchester, Manchester Canc Res Ctr, Manchester, Lancs, England (2018)
    • A simple one-pot preparation of 4-alkoxy and 4-alkylthio-catechols and o-benzoquinones.

      Cooksey, C J; Land, Edward J; Department of Chemistry, University College London, London, UK (1996)
    • Abarelix and other gonadotrophin-releasing hormone antagonists in prostate cancer.

      Kirby, Roger S; Fitzpatrick, John M; Clarke, Noel W; The Prostate Centre, London, UK. (2009-12)
      Hormonal therapy is the main recommended treatment for locally advanced and metastatic prostate cancer. Luteinizing hormone-releasing hormone (LHRH) agonists, such as buserelin, goserelin, leuprorelin and triptorelin, stimulate the pituitary's gonadotrophin-releasing hormone (GnRH) receptor, ultimately leading to its de-sensitization and subsequent reduction of LH and testosterone levels. However, this reduction is accompanied by a well described increase or 'surge' in LH and testosterone levels, necessitating the concomitant administration of an antiandrogen to combat the potential effects of transient acceleration in cancer activity. Two pure GnRH antagonists have been developed, abarelix and degarelix, that are devoid of any agonist effect on the GnRH receptor and consequently do not result in testosterone flare. Abarelix was the first GnRH antagonist to be developed and was approved by the USA Food and Drug Administration in 2004 for the initiation of hormonal castration in advanced or metastasizing hormone-dependent prostate carcinoma, when rapid androgen suppression is necessary. Clinical data on both abarelix and degarelix show that they can produce rapid and sustained decreases in testosterone to castrate levels without the need for co-administration of an antiandrogen, and with a very low complication rate in the short term.
    • Abatacept acute graft-versus-host disease prophylaxis is feasible and safe when combined with post-transplant cyclophosphamide in the mobilised peripheral blood haploidentical donor setting

      Yong, J.; Patel, Alkesh; Haematology, Clatterbridge Cancer Centre, Liverpool (2020)
      Acute graft-versus-host disease (aGVHD) remains a major limitation to allogeneic haematopoietic stem cell transplantation (HSCT), associated with high patient morbidity and mortality. Steroid refractory acute GVHD (SR-aGVHD) has the highest mortality (around 80%) despite treatment intensi'cation with available immunosuppressive therapy. This is complicated by poor response rates alongside increased toxicity and infectious complications from profound immunosuppression and prolonged uncontrolled GVHD. Novel immunotherapeutic strategies are therefore urgently needed for treatment of SR-aGVHD. Immunological manipulation of T-cell activation is a novel therapeutic treatment approach for aGVHD. Abatacept, a CTLA4-Ig exerting CD28-CD80/CD86 axis T-cell co-stimulation blockade, has been shown in the phase 1 clinical trial setting to be safe, well tolerated without dose limiting toxicities (DLTs), and ef'cacious in the treatment of heavily pretreated chronic extensive GVHD (cGVHD). Marked improvement in National Institutes of Health cGVHD scores alongside signi'cantly reduced steroid dose requirement in this cohort have led to a phase 2 trial (NCT01954979) and National Comprehensive Cancer Network (NCCN) recommendation. T-cell activation is a key component of aGvHD but it is currently unknown if Abatacept represents a potential therapy for SR-aGVHD. We report the feasibility, safety and ef'cacy of Abatacept added to the management of SR-aGVHD in a cohort of four heavily pretreated patients in a single centre. Institutional compassionate access was granted after exhaustion of all available therapies. Three of the four patients developed aGvHD after donor lymphocyte infusion (DLI), in the absence of pre-DLI conditioning or GVHD prophylaxis. Median overall MAGIC aGVHD score was 4, with two patients having predominant stage 4 gut aGVHD and two patients with predominant stage 4 skin aGVHD. The median prior lines of treatment were 5, including steroid therapy (Prednisolone or Methylprednisolone 2 mg/kg/day), calcineurin or mTOR inhibitor, mycophenolic acid and extracorporeal photophoresis (ECP) with 8methoxypsoralen (8-MOP). Patients with gut aGVHD were also treated with vedolizumab and/or in'iximab. Abatacept 10 mg/kg was administered IV in addition to concurrent treatments for SR-aGVHD at d0, d+14, d+28, d+56, d+72 and d+90. If a clinical response was observed after 6 doses, Abatacept was continued every 4'6 weeks up to 12 doses. Two patients achieved a clinical partial response and one patient a clinical complete response at d+100 after initiation of Abatacept. Importantly all patients achieved a median dose reduction in steroids of 98% by d+100. Three patients had viral reactivation diagnosed prior to Abatacept initiation (median of d'155 before Abatacept). Importantly, no new viral reactivations were detected in any patients after Abatacept initiation. No DLTs were observed; bacterial infections were not considered a DLT if they had already occurred with prior therapies, being attributed to pre-existing severe immunosuppression and SRaGVHD. We observed that patients with less than CR by d+100 developed chronic GVHD of the involved organ. Mortality was observed only in the two patients with gut involvement, as a result of chronic malnutrition and recurrent immunocompromised infection. Our experience suggests that Abatacept for SR-aGvHD is feasible, appears safe and may have ef'cacy in some patients. A prospective clinical trial is planned.
    • Aberrant activation of notch signaling in human breast cancer.

      Stylianou, Spyros; Clarke, Robert B; Brennan, Keith; Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Christie Hospital NHS Trust, Manchester M13 9PT, United Kingdom. (2006-02-01)
      A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.
    • Aberrant CDKN1A transcriptional response associates with abnormal sensitivity to radiation treatment.

      Badie, Christophe; Dziwura, S; Raffy, C; Tsigani, Theodora; Alsbeih, G; Moody, J; Finnon, Paul; Levine, Edward; Scott, David A; Bouffler, Simon; et al. (2008-06-03)
      Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2 h following 2 Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (P<0.002) in severe reactors compared to normal. Prediction of reaction severity of approximately 91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests.
    • Ability to undergo apoptosis does not correlate with the intrinsic radiosensitivity (SF2) of human cervix tumor cell lines.

      Sheridan, Mary T; West, Catharine M L; CRC Experimental Radiation Oncology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (2001-06-01)
      PURPOSE: To investigate the relationship between radiation-induced apoptosis and clonogenic cell kill in 9 cervical cancer cell lines. METHODS AND MATERIALS: Cells were irradiated with 0, 2, 8, and 30 Gy. The level of apoptosis was evaluated using flow cytometry (Annexin-V binding), light microscropy (morphology), gel electrophoresis (DNA ladder formation), and TUNEL assay. Cell survival was measured using a clonogenic assay. RESULTS: Of the 9 cervical carcinoma cell lines analyzed, 3 underwent radiation-induced apoptosis: CaSki, HT3, and 778. The levels of apoptosis, obtained 72 h after a dose of 30 Gy, were 49%, 28%, and 26%, respectively. All cell lines exhibited some level of background apoptosis measured by Annexin-V binding (mean = 2.6%+/-0.8; range, 0.2-6.9%) that correlated with the level of radiation-induced apoptosis (r = 0.92, p = 0.001). In 6 of the 9 lines, necrosis was the dominant form of cell death. A significant inverse relationship was found between the level of radiation-induced apoptosis and necrosis after 30 Gy (r = -0.87, p = 0.002). No relationship was found between radiation-induced apoptosis and intrinsic radiosensitivity measured, using a clonogenic assay, as surviving fraction at 2 Gy (SF2). CONCLUSION: Cervical carcinoma cells do not readily undergo radiation-induced apoptosis in vitro. There is no relationship between ability to undergo apoptosis and intrinsic radiosensitivity measured using a clonogenic assay.
    • Abl protein kinase abrogates the response of multipotent haemopoietic cells to the growth inhibitor macrophage inflammatory protein-1 alpha.

      Wark, G; Heyworth, Clare M; Spooncer, Elaine; Czaplewski, L; Francis, Julia M; Dexter, T Michael; Whetton, Anthony D; Leukaemia Research Fund Cellular Development Unit, UMIST, Manchester, UK. (1998-03-12)
      The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic progenitor cells, are resistant to the growth inhibitory effects of the chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha). CML is also relatively resistant to chemotherapy and the disease is difficult to cure using conventional therapeutic routes. CML is associated with increased abl oncogene protein tyrosine kinase (PTK) activity. Here, we have tested the hypothesis that these aberrant responses to MIP-1alpha and the relative resistance to chemotherapy are directly related to this increased abl PTK activity in primitive haemopoietic cells. To do this we have expressed a temperature sensitive abl PTK in a growth factor dependent, multipotent stem cell line (FDCP-Mix) in which growth is normally suppressed by MIP-1alpha. In FDCP-Mix cells expressing the ts v-abl PTK and grown at the restrictive temperature for PTK activity the cells were relatively sensitive to cytotoxic agents such as cytosine arabinoside and 5-fluorouracil but MIP-1alpha could induce growth inhibition and confer some degree of protection from these agents. At the permissive temperature for abl PTK, the cells were relatively resistant to cytotoxic drugs and MIP-1alpha treatment neither induced growth inhibition nor protected the cells from cytotoxic drug induced cell death. This lack of response to MIP-1alpha was not due to receptor down modulation as neither the affinity nor the number of 125I-MIP-1alpha binding sites was altered by activating Abl PTK. However, MIP-1alpha mediated increases in cytosolic Ca2+ levels were abrogated by switching cells to the permissive temperature for Abl PTK activity. These data suggest that the relative resistance of CML progenitor cells to therapeutic drugs and the lack of response to MIP-1alpha occurs as a direct consequence of abl PTK activity and involves desensitisation of signal transduction events stimulated by MIP-1alpha receptors. Thus one contributory mechanism to transformation of primitive haemopoietic cells is abrogation of response to a growth inhibitor.
    • Ablation of murine jejunal crypts by alkylating agents.

      Moore, James V; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-02)
      The gut microcolony assay has been used to measure damage to intestinal crypts by single and split doses of 3 alkylating agents: mechlorethamine hydrochloride (HN2), bis-chloroethyl-nitrosourea (BCNU) and isopropyl methane sulphonate (IMS). The single-dose survival curves for whole crypts were distinguished by extrapolation numbers (3.0, 176 and 1.5 respectively) that were lower than most previously published values for assay by irradiation. Significant sparing of crypts occurred when doses of HN2 or BCNU, but not IMS, were given in 2 equal fractions separated by more than 2 h. Deduced D0 values for those cells from which crypts regenerate were 1.9 mg/kg HN2, 19 mg/kg BCNU and 487 mg/kg IMS.
    • The abnormal cytotoxicities of 2,5-diaziridinyl-1,4-benzoquinone-3-phenyl esters.

      Di Francesco, A; Hargreaves, R; Wallace, T; Mayalarp, Stephen P; Hazrati, A; Hartley, J; Butler, John; Department of Biological Sciences, Salford University, UK. (2000-10)
      Several derivatives of 2,5-diaziridinyl-3-phenyl-1,4-benzoquinone have been synthesized and their cytotoxicities in six different human cancer cell lines (H460, H596, HT29, BE, K562 and A2780) have been determined. It was observed that certain phenol-ester derivatives were significantly more cytotoxic in all of the cell lines investigated. These esters were shown to be cleaved by esterases to form a stable meta-phenol and an unstable para-phenol. The meta-phenol was also highly cytotoxic. Several of these compounds were studied in detail using DNA cross-linking, clonogenic, apoptosis and flow cytometry assays. It is proposed that although the phenol-esters and the phenols can efficiently cross-link DNA, this mechanism alone is not sufficient to explain the toxicities of these compounds.
    • Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients.

      Betticher, Daniel C; Heighway, Jim; Thatcher, Nick; Hasleton, Philip S; CRC Department of Med. Oncology, Christie Hospital (NHS) Trust, Manchester, UK. (1997)
      Tumours develop through the accumulation of genetic alterations associated with a progressive increase of the malignant phenotype. In lung cancer, chronic exposure of bronchial epithelium to carcinogens in cigarette smoke may lead to multiple dysplastic and hyperplastic lesions scattered throughout the tracheobronchial tree. Little is known about the genetic alterations in such lesions. This study was carried out to examine cyclin D1 (CCND1) and retinoblastoma (RB1) gene expression in the bronchial epithelium of patients with lung cancer. Lung tumours and their corresponding tumour-free resection margins from 33 patients who underwent resection of non-small-cell lung cancer (NSCLC) were examined by immunostaining with monoclonal antibodies against cyclin D1 (DCS-6; Novocastra) and pRb (NCL Rb-1; Novocastra). Examination of the resection margins revealed four carcinomas in situ, 19 hyperplasias and ten sections showing apparently normal bronchial epithelium. A control group of patients, without lung tumours and who had never smoked, revealed no or weak cyclin D1 and positive pRb staining within bronchial epithelia. Increased cyclin D1 and diminished pRb expression were found in 76% (n = 25) and 27% (n = 9) of the resection margins respectively, and in 12% (n = 4) both cyclin D1 and pRb expression were altered. In the corresponding tumours, 48% (n = 16) were normal, while altered expression was found for cyclin D1 in 33% (n = 11), pRb in 27% (n = 9) and both in 9% (n = 3) of cases. It appears that altered expression of cyclin D1 and pRb is an early event in NSCLC development in almost half of cases analysed. Further investigations are needed to determine the significance of immunostaining of bronchial specimens in individuals at risk of lung cancer, with the possibility that the observations are of importance in the early diagnosis of NSCLC.
    • Abnormal intracellular distribution of O6-alkylguanine-DNA-alkyltransferase in hepatitis B cirrhotic human liver: a potential cofactor in the development of hepatocellular carcinoma.

      Lee, Siow Ming; Portmann, B C; Margison, Geoffrey P; CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK. (1996-11)
      The expression of O6-alkylguanine-DNA-alkyltransferase (ATase), which is responsible for repair of the promutagenic and cytotoxic DNA lesion O6-alkylguanine, was examined by immunostaining in a series of liver sections from normal and hepatitis-B cirrhosis patients using a polyclonal anti-human ATase antiserum. In 10 normal liver sections, the ATase staining was predominantly nuclear and very intense in the majority of the hepatocytes with a panacinar distribution. In contrast, in 15 hepatitis-B sections, the ATase was located mainly in the cytoplasm of the majority of the hepatocytes and had a perinuclear distribution. Scattered hepatocytes showed a more intense staining involving all or part of their cytoplasm; nuclear staining, however, was reduced in intensity, being inconspicuous in seven and patchy and weak in eight. ATase activity in extracts of 10 of the hepatitis-B livers varied from approximately 90 to 360 fmoles/mg protein and there was no apparent relationship between these levels and the cellular distribution of the enzyme. The sequestration of the ATase protein in the cytoplasm, away from its site of action in the cell nucleus suggests that in hepatitis-B cirrhosis, the repair of O6-alkylguanine lesions by ATase may be less efficient than in normal hepatocytes. The abnormal cellular distribution of ATase may thus be an additional cofactor in the development of hepatitis-B-associated hepatocellular carcinoma.