British Journal of Cancer

Epstein, RJ; Smith, PJ; Watson, JV; Bleehen, NM

doi: 10.1038/bjc.1988.104pmid: 3395549

Cycling cells are recognised to be more susceptible than quiescent cells to the cytotoxic action of many commonly used cancer chemotherapeutic agents. We have found that oestrogen stimulation of T-47D human breast cancer cells is accompanied by a two-fold increase in VP-16-induced DNA cleavage as measured by alkaline DNA unwinding, and that this increase in DNA cleavage is accompanied by a corresponding enhancement of drug-induced cytostasis. The enhancement of VP-16-induced DNA cleavage seen with oestrogen exposure is antagonised both by antioestrogen treatment and by cycloheximide, an inhibitor of protein synthesis, but not by the DNA synthesis inhibitor aphidicolin. Increased c-myc protein synthesis is detectable within an hour of oestrogen exposure, while increased VP-16-induced DNA cleavage is detectable within 4h and increased DNA synthesis within 16h. Only small changes in cell-cycle distribution occur with oestrogen stimulation. In the absence of VP-16, oestrogen does not reduce DNA double-strandedness, nor does it induce changes in chromatin structure as measured by alterations in DNA superhelicity or chromatin accessibility. These findings suggest that oestrogen enhances VP-16-induced DNA damage in asynchronously growing G1-phase cells and that this enhancement may be dependent at some point upon de novo protein synthesis. Oestrogen pre-treatment of T-47D human breast cancer cells improves the therapeutic index of VP-16 without the need for cell synchronisation or highly precise drug scheduling.

Benstead, K; Moore, JV

doi: 10.1038/bjc.1988.105pmid: 3395550

The clearance of an intradermally-injected solution of 133Xenon in 0.9% saline has been used to study the impairment and recovery of blood flow in mouse tail for 5 days following photodynamic therapy (PDT) with 2mg TPPS i.v. per mouse and a range of doses of white light. Impairment of blood flow was observed within 10 min of light exposure. Blood flow increased between day 1 and day 5 at light doses less than 151J cm-2 and had returned to control levels by day 5 at light doses less than 129J cm-2. In mice treated with a light dose that caused a 50% incidence of necrosis, there was no significant difference in the initial xenon clearance half-time (measured at 10 min and 1 day after PDT) between those mice which developed tail necrosis and those which healed. However, the latter showed significantly greater improvement in vascular function on days 2, 3 and 4. This suggests that the timing and extent of recovery of blood flow determined the risk of necrosis in individual mice.

White, L; Gomer, CJ; Doiron, DR; Szirth, BC

doi: 10.1038/bjc.1988.106pmid: 3395551

Haematoporphyrin derivative (HPD) photodynamic therapy (PDT) may have clinical application in the management of patients with retinoblastoma. Heterotransplantation of retinoblastoma cells into the anterior chamber of the nude mouse eye and the subsequent growth of small tumour masses has provided a model for evaluation of various therapeutic modalities. Ninety-four evaluable xenograft tumours in 54 nude mice were randomized to receive one of the following treatments: cyclophosphamide (CPM) alone, HPD-PDT alone, CPM followed by HPD-PDT, HPD-PDT followed by CPM, or saline control. Responses were demonstrated after CPM treatment in all three relevant groups. However, HPD-PDT was found to be ineffective either alone or as a contributor in the double modality treatment groups. The small tumour masses treated can be demonstrated histologically to be avascular. It is proposed that although the same retinoblastoma cells in different circumstances are responsive to HPD-PDT, no clinical response is demonstrable utilizing this model, due to the absence of tumor vascularity.

Hughes, JM; deFazio, A; Tattersall, MHN

doi: 10.1038/bjc.1988.107pmid: 3395552

A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.

Biunno, I; Pozzi, MR; Pierotti, MA; Pilotti, S; Cattoretti, G; Della Porta, G

doi: 10.1038/bjc.1988.108pmid: 3293644

We have performed an analysis of ras, c-myc, c-myb, c-erbB1 and c-erbB2 oncogenes in 100 surgical samples of human breast carcinomas. No point mutations have been detected at the 12th codon of c-Ha-ras and c-Ki-ras in 40 and 65 breast cancer DNAs, respectively. One out of 65 samples showed a 50-fold amplification of c-Ha-ras that, however, was not overexpressed. Alterations in the structure of c-myc, c-myb c-erbB1 and c-erbB2 oncogenes were sporadically observed. In 20 tumour samples, the study of expression of a series of oncogenes revealed that c-Ha-ras was the predominantly transcribed gene among the ras gene family whereas c-fos appeared the most constantly and significantly expressed nuclear oncogene.

Antoine, E; Pauwels, C; Verrelle, P; Lascaux, V; Poupon, MF

doi: 10.1038/bjc.1988.109pmid: 2969255

Rats bearing a transplanted nickel-induced rhabdomyosarcoma (RMS 9-4/0), treated with chlorozotocin (CZT), an alkylating agent, showed an amplified metastatic invasion of the lung (median of 165 lung tumour nodules, compared to 3 for untreated controls). A higher level of metastatic invasion (200 nodules) was reached spontaneously after the grafting of the S4T line, which was obtained by successive in vivo passages of RMS 9-4/0 cells in CZT treated rats. S4T tumour cells also invaded the liver and a considerable proportion of the lymph nodes. The NT4T line, obtained by successive in vivo passages in untreated rats, showed a lesser degree of enhancement of metastatic capacity (57 nodules). Both derived lines proved to be more aggressive than the parental, proliferated more rapidly, and were resistant to CZT toxicity. Only the non-treated lineage became more resistant to NK lysis. The S4T line lost its myogenic differentiation and was best described as a fibrohistiosarcoma, whereas NT4T did not. Chromosome analysis demonstrated a reduced range of chromosome number per cell in both lines. We conclude that both S4T and NT4T tumours became more metastatic than RMS 9-4/0 as the result of tumour progression through in vivo passages, and that in addition S4T acquired a spontaneously higher metastatic potential, similar to that which occurred in rats grafted with RMS 9-4/0 or NT4T tumours and treated by CZT. This suggests an inheritable mutation in the S4T line.

Teale, DM; Khidair, IA; Potter, CW; Rees, RC

doi: 10.1038/bjc.1988.110pmid: 2840108

The effect of basement membrane components (laminin, fibronectin and type IV collagen) and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced.

D'Onofrio, C; Perno, CF; Mazzetti, P; Graziani, G; Calio', R; Bonmassar, E

doi: 10.1038/bjc.1988.111pmid: 2899440

Natural human interferon beta (beta-IFN) was tested during the early phase of in vitro infection with HTLV-I virus of human cord blood mononuclear cells (CBL), to evaluate whether its antiviral and immunomodulating effects might prevent spreading of infection in the host. beta-IFN was found to reduce HTLV-I transmission and integration in CBL cultures. Moreover, beta-IFN had no effect in preventing virus transmission and integration in K562 and a very limited effect in HL60 and Molt-4 human tumour lines, suggesting a cell-type specific mode of action. beta-IFN induced a 'priming' response on CBL, since overnight pretreatment of recipient cells or one single treatment at the onset of the coculture were almost equally effective in protecting against HTLV-I infection. During the early days post infection (p.i.), IFN-treated CBL showed a pattern of phenotypic markers that was closer to that of non-infected CBL. In contrast, untreated CBL exposed to HTLV-I showed a percent increase of Tac+, M3+ and Leu 11+ subpopulations. Cell-mediated immune responses of CBL were depressed after coculturing with HTLV-I producer MT-2 cells. beta-IFN was able to boost the cell-mediated cytotoxicity of fresh and infected CBL against both K562 and MT-2 target cells. Leukocyte blastogenesis in mixed lymphocyte/tumour cell cultures, evaluated in terms of 3H-thymidine incorporation during the first week p.i., was also enhanced by IFN when macrophages and lymphocytes were reconstituted at an optimal 1:20 ratio. It is conceivable that this overall enhancement of the immune response induced by beta-IFN could contribute to reduce HTLV-I infection in vitro.

Foxwell, BMJ; Band, HA; Long, J; Jeffery, WA; Snook, D; Thorpe, PE; Watson, G; Parker, PJ; Epenetos, AA; Creighton, AM

doi: 10.1038/bjc.1988.112pmid: 3395553

In recent years, radiolabelled monoclonal antibodies have been evaluated for their use in the diagnosis and treatment of neoplastic disease. One isotope which has not been assessed for antibody targeting is 32P, even though it has many favourable radiobiological characteristics and has been used clinically for the treatment of certain neoplastic disorders such as polycythaemia rubra vera. The main drawback so far in using 32P has been the absence of a general method for phosphorylating antibodies. We have now developed a novel process for the phosphorylation of immunoglobulins which is rapid, efficient and allows high specific activities to be achieved (greater than 10 muCi micrograms-1). The technique involves the chemical conjugation of Kemptide, a synthetic heptapeptide substrate for kinases, to immunoglobulins. The antibody-Kemptide conjugate can then be phosphorylated using protein kinases and [32P]-gamma-ATP. The procedure does not compromise the binding activity of the antibody. The 32P-labelled monoclonal antibodies were stable in human, mouse and rat plasmas in vitro, although they cleared from the bloodstream of mice with a beta-phase half life of 2 days which is approximately two times faster than that of native antibody. The application of this phosphorylation technique should allow the therapeutic potential of targeted 32P to be assessed.

Rofstad, EK; Fenton, BM; Sutherland, RM

doi: 10.1038/bjc.1988.113pmid: 3395554

Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen consumption and cell survival time under hypoxic stress.