Selective interactions of verapamil with anthraquinones in adriamycin-sensitive and-resistant murine and human tumour cell lines in vitro

Summary Enhancement of the cytotoxicity of adriamycin (ADR) by addition of verpamil (VPM) was selective and, in part, concentration-dependent. In an ADR-resistant murine L5178Y lymphoma subline sensitivity was improved with ADR and 1 M VPM, whereas the cytotoxic effects of mitoxantrone or esorubicin were not affected by VPM. In human ovarian SK-OV-3 tumour cells ADR cytotoxicity was only enhanced by 6.6 M VPM and there was no selective advantage against an ADR-resistant subline. Circumvention of ADR resistance by VPM is not a universal phenomenon, appearing least effective against sublines expressing relatively low orders of resistance, which may be those most commonly encountered clinically.     

Effects of 5-fluorouracil,leucovorin, and glucarate in rat colon-tumor explants

Summary In a previous study, we showed that 5-fluorouracil (FU) is active against the dimethylhydrazine-induced colon tumor in rats; a 7-day infusion of FU at 30 mg/kg daily produced 85% tumor-free cures. The present study examined the effects of FU alone and in combination with leucovorin (LV) ord-glucarate (GT) using an ex vivo system that maintained the growth of the rat colon-tumor explants on collagen gels. The labeling index (LI) was determined by the incorporation of [³H]-thymidine and autoradiography. The mean LI of the untreated control was 64.8%±19.8%. The IC₅₀, IC₉₀, and IC₉₅ values following a 7-day exposure to FU were 0.36, 0.75, and 1.22 m, respectively. In comparison, the steady-state FU concentration required to produce 67% tumor-free cures in rats following a 7-day infusion is 1.54 m. LV alone did not produce any antiproliferative effect at concentrations as high as 10 m. The addition of LV at concentrations of 0.001–10 m did not significantly reduce the IC₅₀ of FU. The lack of effect of LV may have been due to tissue saturation with folate provided in the culture medium. GT alone reduced the tumor LI by 20%–30% at concentrations of 0.1–10 m. GT enhanced the effect of FU. As compared with FU alone, the addition of GT at concentrations of 0.1 and 1.0 m reduced the IC₅₀ of FU by 47% and 60% to 0.21 and 0.16 m, respectively. Assessment of the potentiation of the inhibitory effect of FU by GT using two-way analysis of variance and the isobologram method indicated a significant synergistic interaction between FU and GT. This interaction occurred within the FU concentration range of 0.08 and 0.4 m. In summary, these data indicate that (a) the IC values for FU are comparable in tumor explants and in rats, suggesting that the effects in cultured tumors reflect those in intact animals; (b) GT alone showed antitumor activity, albeit relatively minor as compared with FU; (c) FU and GT exhibited synergistic activity, which was most pronounced at FU concentrations that produced submaximal activity (<30% inhibition of tumor LI); and (d) GT and LV had different effects on the growth inhibition by FU, suggesting that GT acts by a mechanism different from the thymidylate synthase-directed effect of FU and LV.Abbreviations FU 5-Fluorouracil - LV leucovorin - GT d-glucarate - LI labeling index - IC inhibitory concentration - IdR thymidine Supported in part by research grants RO1 CA-43365 and RO1 CA-51757 and by Research Career Development Award K04 CA-01 497 (to J. L.-S. A.) from the National Cancer Institute, NIH, DHHS. The work of the senior author (T. D. S.) was supported in part by a fellowship from the Berlex Corporation     

Inhibitory effect of bile salts on the enterohepatic circulation of methotrexate in the unanesthetized rat: Inhibition of methotrexate intestinal absorption

Summary The effect of conjugated and unconjugated bile salts on the intestinal absorption of methotrexate (MTX) in the unanesthetized rat was investigated using a recycling perfusion technique. We initially determined the general characteristics of MTX absorption in vivo. Absorption of low (0.5 M) and high (6 M) concentrations of MTX was linear with time for 60 min perfusion and occurred at rates of 0.2 and 1.65 nmol/100 cm dry length/min, respectively. Absorption of 0.5 M MTX was pH-dependent and increased with decreasing perfusate pH. Absorption of MTX involves two processes: (1) a saturable process with a K_t of 0.98 M, and (2) a nonsaturable diffusion process. The unconjugated deoxycholate and the conjugated taurocholate inhibited the intestinal absorption of 1 M MTX in a concentration-dependent manner. The inhibitory effect of bile salts was reversible, and was not due to damage to the intestinal mucosa. The structural analogues folic acid and 5-methyltetrahydrofolate and the organic anions rose bengal and sulfobormophthalein were also inhibitory to MTX absorption. This study demonstrates that a variety of organic anions inhibit MTX intestinal absorption. The possible therapeutic importance of this observation is discussed.An abstract of this work was presented at the American Gastroenterology Annual Meeting, New Orleans, LA, May, 1984 and appeared in Gastroenterology 86: 1228, 1984Supported by the U.S. Public Health Service Grant AG 2767     

Potentiation of etoposide-induced cytotoxicity and DNA damage in CCRF-CEM cells by pretreatment with non-cytotoxic concentrations of arabinosyl cytosine

Summary Pretreatment of the human lymphoblastoid cell line CCRF-CEM with 0.02 m arabinosyl cytosine (ara C) enhances both the cytotoxic and the DNA-damaging effects of etoposide. This concentration of ara C is itself non-cytotoxic and results in no detectable DNA damage as measured by alkaline elution. Ara C pretreatment results in the synchronisation of cells, a 24-h pretreatment resulting in the accumulation of cells in the early S phase. The sensitivity of cells to etoposide-induced cytotoxicity was increased 2.5 times and DNA damage was enhanced 1.66 times by this pretreatment. Maximal potentiation of etoposide-induced DNA damage (2.06-fold increase) was observed after 48 h continuous treatment with ara C, but no further enhancement of cytotoxicity occurred. Cell-cycle analysis demonstrated that 48 h ara C treatment resulted in the accumulation of cells in the late S/G₂M phase. Cells returned to a normal cell-cycle distribution within 24 h of the removal of ara C, and the potentiation of etoposide activity was then reduced to a 1.3- to 1.4-fold level. DNA damage induced by etoposide following ara C pretreatment was qualitatively identical to that produced by etoposide alone, suggesting a mechanism involving topoisomerase II. To investigate this possibility, we measured topoisomerase II protein levels by immunoblotting. Measurement of topoisomerase II levels in whole-cell lysates of ara C-pretreated cells showed a 3- to 5-fold increase in topoisomerase levels relative to total protein content. This suggests that elevated enzyme levels may be responsible for the increased sensitivity of ara C-pretreated cells to etoposide.Abbreviations ara-C cytosine -d-arabinofuranoside - m-AMSA 4-(9-acridinylamino)-methanesulfon-m-anisidide - DMSO dimethylsulphoxide - MTT 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide - SSBs single-strand breaks This work was supported by a grant from the North East Thames Regional Health Authority     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

1-β-d-arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation

We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1--d-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induced apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced intergenomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in <1.0 g of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 M Ara-C for 4 h, which increased with 10 and 50 M Ara-C. Incubation with 100 M Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 M MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 M MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 M MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 M for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 M) of TXL. Although continuous exposure to 1.0 M TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which non of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.     

Biochemical modulation of 5-fluorouracil with or without leucovorin by a low dose of brequinar in MGH-U1 cells

Summary Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical antitumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU±leucovorin (LV)±brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC₅₀) for brequinar, FU, and FU+LV (100 m) were 0.4, 20, and 10 m, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC₁₀ (0.1 m) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 m brequinar and FU±LV did not increase the cytotoxicity of FU±LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 m brequinar for 24 h alone or followed by a 2-h exposure to FU (25 m)±LV (100 m). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU + brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 m hypoxanthine and 25 m FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).Supported by the National Cancer Institute, Canada, and Du Pont Pharma, Mississauga, Canada     

Retinoid,retinoic acid receptor beta and breast cancer

Retinoids have been reported to inhibit the growth of several breast cancer cell lines in culture and to reduce breast tumor growth in animal models. Furthermore, retinoic acid (RA) can augment the action of other breast cancer cell growth inhibitors both in vitro and in vivo. Clinically, interest has increased in the potential use of retinoids for the prevention and treatment of human breast cancer. The regulation of cell growth and differentiation of normal, premalignant, and malignant cells by retinoids is mediated by the RA receptors (RARs) and retinoid X receptor. One of the target genes of retinoid receptors is RAR2. A growing body of evidence supports the hypotheses that the RAR2 gene is a tumor suppressor gene and the chemopreventive effects of retinoids are due to induction of RAR2. RAR2 expression is reduced in many malignant tumors including breast carcinoma. This paper will briefly discuss basic aspects of retinoids and retinoid acid receptor. In particular, we review what is now known for inactivation mechanism of RAR2 and its role in tumor cell growth inhibition.     

Micromolar concentrations of 2-methoxyestradiol kill glioma cells by an apoptotic mechanism,without destroying their microtubule cytoskeleton

The purpose of this study was to investigate the potential effects of 2-methoxyestradiol, a natural mammalian steroid, in glioma cells, since antiproliferative effects of this compound had been shown earlier in several leukemia and carcinoma cell lines. The effects of 0.2, 2 and 20M concentrations of 2-methoxyestradiol were measured in three malignant human glioma cell lines (U87MG, U138MG, LN405) and one malignant rat glioma cell line (RG-2) using a microtiter-tetrazolium (MTT) assay. In all cell lines, a significant reduction of the viable cell number by more then 75% occurred ( P < 0.05) for concentrations of 2 and 20M 2-methoxyestradiol after 6days. A concentration of 0.2M had smaller effects (10–40% cell reduction), which were significant in two of the cell lines tested. The apoptotic nature of cell death was further analyzed in U87MG and RG-2 cells. Caspase-3 activity was significantly induced to levels between 3.4- and 23-fold after 4days for the two higher 2-methoxyestradiol concentrations (P < 0.05). In the cell line RG-2 nuclear fragmentation was visible in many nuclei, following stains with Hoechst H33258. A round cell morphology occurred in most treated cells, which was not accompanied by a complete destruction of the microtubule network, as it can be observed with other microtubule targeting drugs. K. Chamaon: These authors contributed equally to the work.J. Stojek: These authors contributed equally to the work.     

Nationwide randomized comparative study of daunorubicin and aclarubicin in combination with behenoyl cytosine arabinoside, 6-mercaptopurine,and prednisolone for previously untreated acute myeloid leukemia

Aclarubicin was evaluated in combination chemotherapy for adult acute myeloid leukemia in a randomized trial involving 58 institutions throughout Japan. Behenoyl cytosine arabinoside (BH-AC)daunorubicin, 6-mercaptopurine, and prednisolone (DMP) was compared with BH-ACaclarubicin, 6-mercaptopurine, and prednisolone (AMP). In the 360 evaluable cases among the 433 cases enrolled, complete remission (CR) rates were 63.7% (116/182) for BH-ACDMP and 53.9% (96/178) for BH-ACAMP (P=0.0587). Median survival periods and 7-year survival rates were 15.8 months and 19.3% for BH-ACDMP and 9.5 months and 20.2% for BH-ACAMP (P=0.0091 according to the generalized Wilcoxon test [GW],P=0.196 according the log-rank test [LR]). Median disease-free survival periods were 15.4 months for BH-ACDMP and 14.1 months for BH-ACAMP (P=0.851 by GW,P=0.439 by LR). Among the 346 cases of extramurally confirmed FAB subtypes, CR rates were 67.9% (19/28) with BH-ACDMP and 31.8% (7/22) with BH-ACAMP for subtype M3 (P=0.011) and 63.3% (93/147) with BH-ACDMP and 56.8% (84/148) with BH-ACAMP (P=0.254) for subtypes M1, M2, M4, and M5. Diarrhea, ileus, pneumonia, and renal failure were more frequent with BH-ACAMP than with BH-ACDMP. The results indicate, at least on the basis of the long-term outcome, that BH-ACAMP has antileukemic effects on subtypes M1, M2, M4, and M5 that are comparable with those of BH-ACDMP.This work was supported by a grant-in-aid from the Japanese Foundation for Multidisciplinary Treatment of Cancer (Cooperative Project 2)     

Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-β-d-arabinofuranosyluracil

Summary Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200–1000 m) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 m ara-C following the exposure of cells to ara-U (1mm) resulted in 4.5 pmol ara-C DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 m ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10–100 m) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.Abbreviations ara-C 1--d-arabinofuranosylcytosine - ara-U 1--d-arabinofuranosyluracil - ara-CTP 1--d-arabinofuranosylcytidine triphosphate - HiDAC high-dose ara-C - HiCAU high concentrations of ara-U - dCTP deoxycytidine triphosphate - HiDAU high-dose ara-U - FiTC Fluoroisothiocyanate - dUDP deoxyuridine diphosphate - dUTP deoxyuridine triphosphate - dTTP thymidine triphosphate - BrdUrd bromodeoxyuridine - dCyd kinase deoxycytidine kinase Supported in part by grant CH-35H from the American Cancer Society, by Public Health Service grant CA-12197 from the National Cancer Institute, National Institutes of Health, and by the Gaston Cancer Society     

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma

Summary Fluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5-deoxy-5-fluorouridine (5dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 °C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH₂FH₄), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH₂FH₄, but our results were not always reproducible.This study was supported by grants from Université Paul Sabatier, Conseil Régional Midi-Pyrénées-CNRS and Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés (INSERM)     

Cellular pharmacology of 1-β-d-arabinofuranosylcytosine in human myeloid,B-lymphoid and T-lymphoid leukemic cells

Summary The in vitro inhibitory action and metabolism of 1--d-arabinofuranosylcytosine (ara-C) on human myeloid (HL-60), B-lymphoid (RPMI-8392), and T-lymphoid (Molt-3) leukemic cells was compared. Ara-C produced greater inhibitory effects in Molt-3 cells than in either HL-60 or RPMI-8392 cells. At a 48 h exposure, ara-C was 7 and 10 times more cytotoxic to Molt-3 cells than to HL-60 and RPMI-8392 cells, respectively. The total ara-C uptake to nucleotides and the formation of 1--d-arabinofuranosylcytosine 5-triphosphate (ara-CTP) was about 5 times greater in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The incorporation of ara-C into DNA was also higher in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The mean intracellular half-life of ara-CTP was 31.7, 59.4, and 155 min for RPMI-8392, HL-60, and Molt-3 leukemic cells, respectively. The Km and V_max values of ara-C for deoxycytidine kinase and the feedback inhibition of this enzyme by ara-CTP in the different leukemic cell lines could not explain the differences in metabolism of this analogue in these cells. These data indicate that the increased sensitivity of T-lymphoid leukemic cells to ara-C than as compared with B-lymphoid and myeloid leukemic cells was due to an ireased rate of formation and a longer half-life of ara-CTP in the T-cells.Supported by a grant from the Cancer Research Society. N.O.-P. is a Fellow of the Leukemia Society of America. To whom requests for reprints should be addressed, at Centre de recherche pédiatrique, Hôpital Ste-Justine, 3175 Ch. Côte Ste-Catherine, Montreal, Quebec H3T 1C5, Canada     

Modification of methyliminodiacetato-trans-R,R-1,2-diamminocyclohexane platinum(II) pharmacology using a platinum-specific monoclonal antibody

Summary Platinum complexes are extremely active chemotherapeutic agents. A murine monoclonal antibody designated 1C1 was developed that binds to the thirdgeneration platinum complex methyliminodiacetato-trans-R,R-1,2-diamminocyclohexane platinum(II) (MIDP). Competitive enzyme-linked immunosorbent assay (ELISA) shows that antibody 1C1 binds preferentially to the 1,2-diamminocyclohexane (DACH) side-chain of the platinum complex, although non-DACH-containing platinum complexes can compete for binding at high concentrations. When tested against MCF-7 breast carcinoma cells, the 1C1-MIDP complex caused 50% growth inhibition at 0.63 g Pt/ml, whereas MIDP alone caused 50% growth inhibition at a concentration of 0.16 g Pt/ml. Pharmacokinetic studies in rats using [³H]-MIDP showed that the drug was cleared triphasically from plasma, with elimination-phase half-lives (t_1/2) of 1.2, 10.2, and 243 min for , , and phases, respectively. The MIDP-1C1 complex was cleared with longer half-lives of 5, 26, and 291 min, respectively. The overall clearance rate from plasma of the MIDP-1C1 complex was 10-fold lower than that of MIDP alone (0.37 vs 3.01 ml/kgxmin). Tissue concentrations of [³H]-MIDP 3 h after administration showed that 1C1 antibody prevented MIDP distribution to most organs and dramatically reduced [³H] concentration in the intestine, liver, kidney, heart, and skeletal muscles. Studies are under way to determine the relative therapeutic activity of the 1C1 antibody-MIDP complex and assess whether the 1C1 antibody may be useful for antibodydirected delivery of platinum complexes to tumors.Abbreviations MIDP methyliminodiacetato-trans-R-R-1,2-diamminocyclohexane platinum(II) - MTT 2,5-diphenyltetrazolium bromide thiazolyl blue Supported in part by NIH grant CA 41581 to ARK     

Gastroesophageal reflux disease, use of H2 receptor antagonists, and risk of esophageal and gastric cancer

Objective: The incidence of esophageal adenocarcinoma has risen rapidly in the past two decades, for unknown reasons. The goal of this analysis was to determine whether gastroesophageal reflux disease (GERD) or the medications used to treat it are associated with an increased risk of esophageal or gastric cancer, using data from a large population-based case–control study. Methods: Cases were aged 30–79 years, newly diagnosed with esophageal adenocarcinoma (n=293), esophageal squamous cell carcinoma (n=221), gastric cardia adenocarcinoma (n=261), or non-cardia gastric adenocarcinoma (n=368) in three areas with population-based tumor registries. Controls (n=695) were chosen by random digit dialing and from Health Care Financing Administration rosters. Data were collected using an in-person structured interview. Results: History of gastric ulcer was associated with an increased risk of non-cardia gastric adenocarcinoma (OR 2.1, 95% CI 1.4–3.2). Risk of esophageal adenocarcinoma increased with frequency of GERD symptoms; the odds ratio in those reporting daily symptoms was 5.5 (95% CI 3.2–9.3). Ever having used H₂ blockers was unassociated with esophageal adenocarcinoma risk (OR 0.9, 95% CI 0.5–1.5). The odds ratio was 1.3 (95% CI 0.6–2.8) in long-term (4 or more years) users, but increased to 2.1 (95% CI 0.8–5.6) when use in the 5 years prior to the interview was disregarded. Risk was also modestly increased among users of antacids. Neither GERD symptoms nor use of H₂ blockers or antacids was associated with risk of the other three tumor types. Conclusions: Individuals with long-standing GERD are at increased risk of esophageal adenocarcinoma, whether or not the symptoms are treated with H₂blockers or antacids.     

Expressions of resistance and cross-resistance in teniposide-resistant L1210 cells

Summary Resistance to teniposide (VM-26) by VM-26 selected resistant L1210 cells in culture was attributed to alterations in the flux of VM-26 across the plasma membrane and to functions of homogeneously staining regions that appeared on one or more chromosomes. In the present study, electrophoresis of membrane-cytosol fractions of these resistant sublines demonstrated a protein band, M_r 22 kd, that was not evident in similar fractions of drugsensitive L1210 cells or three revertant sublines. The distribution of this protein among various cellular fractions could be altered by manipulation of the concentration of calcium ions. A representative subline, LIa5 M, was observed to have vesicles that reacted with Sudan black B stain, an indication of altered lipid metabolism. The LIa5 M subline was cross-resistant to etoposide, vincristine, doxorubicin, amsacrine, and actinomycin D. Concentrations of VM-26 that inhibited cell division to the same extent caused an accumulation of fewer cells in the G₂ stage of cell division in LIa5 M cultures than in L1210 cultures. These observations indicate that the LIa5 M subline expressed multiple drug resistance, as well as changes in the expression of cytotoxicity to VM-26.This investigation was supported by Research Project Grant CA 35319, by Cancer Center Support (CORE) Grant CA 21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities.Recipient of research support from a training grant, American Cancer Society Grant IN-85-06, to the University of Tennessee Center for the Health Sciences     

Stereoselective metabolism of ftorafur (R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil)

Summary The metabolism of R and S isomers of ftorafur (FT) was studied in vivo and in vitro. FT and its metabolites, i.e., hydroxylated derivatives, (OH-FT), -butyrolactone (GBL), 5-fluorouracil (FU), and dehydro-FT, were analyzed by gas chromatography and high-pressure liquid chromatography. Although previous results did not demonstrate any differences in the biological activity of the FT isomers, R- and S-FT were metabolized to different extents by individual pathways. Cleavage of R-FT at the N₁-C₂ position to form GBL by non-microsomal soluble enzymes in mouse and rabbit liver homogenates was greater than that of S-FT. Following IP administration of S-FT, both cis- and trans-4-OH-FT were recovered in 24-h rat urine; however, significantly less trans-4-OH-FT and no cis-4-OH-FT was detected following R_FT administration. There were no glucuronide or sulfate conjugates of these OH-FT metabolites. These results indicate that urinary hydroxylated metabolites generated from racemic FT consist predominantly of -l-4-OH-FT and -l-4-OH-FT, and only a small fraction of the trans-4-OH-FT appears in the -d configuration of natural nucleosides, confirming earlier reports. The low extent of urinary excretion of hydroxylated FT metabolites in the -configuration suggests that either there is stereoselective hydroxylation of S-FT or the hydroxylated metabolites with the natural -d configuration generated from R-FT were preferentially further metabolized prior to excretion into urine. A sample of chemically synthesized -d-4-OH-FT was quantitatively converted to FU by thymidine phosphorylase in vitro; this compound may represent a potential FU prodrug.     

Nitrogen mustard-DNA interaction in melphalan-resistant mammary carcinoma cells with elevated intracellular glutathione and glutathione-S-transferase activity

Summary We examined the relationship between intracellular levels of glutathione (GSH), glutathione-S-transferase (GST) activity, and the kinetics of DNA cross-links induced by the bifunctional alkylating drugs melphalan (MLN), chlorambucil (CLB), and mechlorethamine (HN2) in a rat mammary carcinoma cell line (WT) and in a subline selected in vitro for primary resistance to MLN (MLNr, 16-fold resistance). MLNr cells exhibit a 2-fold increase in intracellular GSH concentration and an approximately 5-fold increase in GST activity as compared with the parent cells. They are cross-resistant to a variety of drugs, including CLB (6-fold) and HN2 (14-fold). Treatment of WT cells with 30 m MLN or CLB induced a significant accumulation of DNA-DNA cross-links for up to 8 h, which decreased over a 24-h period. In MLNr cells, no significant cross-link formation was induced by either MLN of CLB at any time between 0 and 24 h. Doses of up to 100 m MLN failed to induce cross-links in MLNr cells. Formation of cross-links was observed immediately after treatment with HN2 in both cell lines and was followed by a subsequent decrease during a 24-h incubation in drug-free medium. At an equimolar concentration (30 m), the mumbers of HN2-induced cross-links were significantly lower in MLNr cells than in WT cells. However, treatment of MLNr cells with 60 m HN2 resulted in cross-link levels similar to those obtained using 30 m HN2 in WT cells. The 35% decrease in MLN accumulation observed in MLNr cells could not entirely explain the absence of crosslinks, since thin-layer chromatographic analysis demonstrated that both cell lines accumulate a significant amount of MLN and metabolize it to the same extent. Significant amounts of MLN were also detected in nuclei isolated from WT and MLNr cells that had been treated with 30 m [¹⁴C]-MLN. Intracellular depletion of GSH by a nontoxic concentration ofl-buthionine-(S, R)-sulfoximine (BSO, 100 m; about 70% GSH depletion) significantly sentisized MLNr cells to MLN and increased cross-link formation. A nontoxic concentration (50 m) of ethacrynic acid (EA, an inhibitor of GST showing some specificity for Yc/Yp subunits) also sensitized MLNr cells to MLN and increased cross-link formation. Our data demonstrate that both EA and BSO are effective modulators of nitrogen mustard cytotoxicity in tumor cells resistant to alkylating drugs. The limited number of cross-links formed in MLNr cells after treatment with MLN or even CLB suggests that efficient repair of drug-DNA monoadducts is operative in these cells and that the increases obtained in the presence of BSO and EA may be related to the involvement of both GSH and GST in drug-DNA interactions such as monoadduct repair.Abbreviations MLN melphalan - CLB chlorambucil - HN2 mechlorethamine - EA ethacrynic acid - BSO l-buthionine-(S,R)-sulfoximine - GSH glutathione - GST glutathione-S-transferase - BCNU Carmustine - CDDP cis-diamminedichloroplatinum(II) This work was supported by research grants from the Cancer Research Society and the National Cancer Institute of Canada     

Progression of Premalignant MCF10AT Generates Heterogeneous Malignant Variants with Characteristic Histologic Types and Immunohistochemical Markers

The purpose of this study was to evaluate the pain experience of women during mammography for breast cancer screening. Possible associations with personal and medical history, sociodemographics and/or situational factors were studied. It was also investigated whether this pain influenced the intention to return for future breast cancer screening. In the Netherlands, women between 50–75 years are invited for screening every two years. A total of 1200 participants were asked to fill up a questionnaire. The response rate was 79.5% (n=954), and 945 questionnaires contained adequate information for analyses. A total of 689 women (72.9%) described mammography as mild to severely painful. In this group, compared to the group that reported no pain, the following factors occurred significantly more often: sensitive breasts (P=0.001), family history of breast diseases (P=0.017), expected pain based on former mammography (P=0.001), high education (P=0.008), anxiety (P=0.001), breast sensitivity in last three days (P=0.001), insufficient attention of technologist (P=0.001). Other factors like age, hormonal status, breast size and hormone use were not associated with the pain experienced. Thirty-two women (3.3%) indicated that they would not attend further screening, 25 (2.6%) reported that the pain might deter them, six women (0.6%) had other reasons, one woman (0.1%) was sure not to come because of severe pain. In conclusion, a large majority of women attending breast cancer screening describes mammography as painful (72.9%). Factors associated with pain were described. Relatively few women (2.7%) indicated that the pain might deter them from future mammography. Recommendations are given to reduce the pain experienced during screening mammography.     

The growth-inhibitory effect of 4-hydroperoxycyclophosphamide against human non-small cell lung carcinoma is enhanced by low-dose difluoromethylornithine

Summary Surgically unresectable buman non-small cell lung carcinoma (NSCC) is highly resistant to present chemotherpy and radiation therapy regimens. Cyclophosphamide, a potent alkylating agent, has shown some efficacy, especially in combination chemotherapy. Difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC) which produces minimal toxicity in animals and humans, has shown antiproliferative effect against human SCC in culture but a much smaller effect (cytostatic) against NSCC. We therefore investigated 4-hydroperoxycyclophosphamide (4HC) and DFMO alone and in combination against a human NSCC line (NCI-H157). Cells were treated with DFMO at graded concentrations of 0 to 800 M from day 0 to day 7. On day 3, cells were exposed for 1 h to 4HC at graded concentrations of 0 to 80 M, washed, and refed with media containing DFMO at initial concentrations. On day 7, cells were counted by hemacytometer. Cells treated with DFMO or 4HC alone exhibited dose-dependent growth inhibition. Growth inhibition by 4HC was enhanced through combination with DFMO. On day 7, 50 M (5×10^-5 M) DFMO effected a 37% inhibition, 8 M 4HC 47% inhibition, and the combination of 50 M DFMO and 8 M 4HC yielded an elevated 71% inhibition. The growth inhibitory effect and potentiating effect of DFMO were reversible upon addition of putrescine (PU) to the culture medium. The combination of DFMO and 4HC, two agents with different toxicity spectra, may represent an effective chemotherapeutic regimen for the treatment of lung cancer.Abbreviations DFMO difluoromethylornithine - FBS fetal bovine serum - 4HC 4-hydroperoxycyclophosphamide - NSCC non-small celllung carcinoma - ODC ornithine decarboxylase - PBS phosphate-buffered saline - PU putrescine - RPMI 1640 Roswell Park Memorial Institute 1640 medium - SCC small cell lung carcinoma The studies were supported in part by grants RO1 CA-43280 and VO1 CA-37606 from the National Institutes of Health. Gordon D. Luk is a recipient of a Faculty Research Award from the American Cancer Society