phosphamide, vincristine, and prednisone. Cancer 56:2369-2375, 1985 (4) KELLER JW, KNOSPE WH, RANEY M, ET AL:
Treatment of chronic lymphocytic leukemia using chlorambucil and prednisone with or without cycle-active consolidation chemotherapy. A Southeastern Cancer Study Group trial. Cancer 58:1185-1192, 1986 (5) BINET JL, LEPORRIER M, DIGHIERO G, ET AL: A
clinical staging system for chronic lymphocytic leukemia: Prognostic significance. Cancer 40:855-864, 1977 (6) LIEPMAN M, VOTAW ML: The treatment of
chronic lymphocytic leukemia with COP chemotherapy. Cancer 41:1664-1669, 1978 (7) KEMPIN S, LEE B r III, THALER HT, ET AL:
Combination chemotherapy of advanced chronic lymphocytic leukemia: The M-2 protocol (vincristine, BCNU, cyclophosphamide, melphalan, and prednisone). Blood 60:11101121, 1982 (8) KEATING Ml, SCOUROS M, MURPHY S, ET AL:
The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy. Blood 57:406-417, 1981 (23) Ho AD, GANESHAGURU K, KNAUF WU, ET AL:
Clinical response to deoxycoformycin in chronic lymphoid neoplasms and biochemical changes in circulating malignant cells in vivo. Blood 6:1884-1890, 1988 (24) PIRO LD, CARRERA CJ, BEUTLER E, ET AL:
2'-Chlorodeoxyadenosine: An effective new agent for the treatment of chronic lymphocytic leukemia. Blood 72:1069-1073, 1988 (25) GREVER MR, KOPECKY KJ, COLTMAN CA,
ET AL: Fludarabine monophosphate: A potentially useful agent in chronic lymphocytic leukemia. Nouv Rev Fr Hematol 30:457-459, 1988 (26) KEATING MJ, KANTARJIAN H.TALPAZM, ETAL:
Fludarabine: A new agent with major activity against chronic lymphocytic leukemia. Blood 74:19-25,1989 (27) O'DWYER PJ, SPIERS ASD, MARSONI S: Asso-
ciation of severe and fatal infections and treatment with pentostatin. Cancer Treat 70: 1117-1120, 1986
(10) FRENCH COOPERATIVE GROUP ON CHRONIC LYM-
PHOCYTIC LEUKAEMIA: Long-term results of the
CHOP regimen in stage C chronic lymphocytic leukaemia. Br J Haematol 73:334-340, 1989 (//) O'DWYER PJ, WAGNER B, LEYLAND-JONES B,
ET AL: 2'-Deoxycoformycin (pentostatin) for lymphoid malignancies. Rational development of an active new drug. Ann Intern Med 108:733-743, 1988 (12) KRAUT EH, BOURONCLE BA, GREVER MR:
Low-dose deoxycoformycin in the treatment of hairy cell leukemia. Blood 68:1119-1122, 1986 (13) SPIERS ASD, MOORE D, CASSILETH PA, ET AL:
Remissions in hairy-cell leukemia with pentostatin (2'-deoxycoformycin). N Engl J Med 316:825-830, 1987 (14) Ho AD, THALER J, MANDELLJ F, ET AL: Re-
sponse to pentostatin in hairy-cell leukemia refractory to interferon-alpha. The European Organization for Research and Treatment of Cancer Leukemia Cooperative Group. J Clin Oncol 10:1533-1538, 1989 (15) MILLER AB, HOOGSTRATEN B, STAQUET M,
ET AL: Reporting results of cancer treatment. Cancer47:207-214, 1981
Metabolic Activation of 4-Ipomeanol in Human Lung, Primary Pulmonary Carcinomas, and Established Human Pulmonary Carcinoma Cell Lines Theodore L. McLemore* Charles L. Litterst, Bruno P. Coudert, Mark C. Liu, Walter C. Hubbard, Steven Adelberg, Maciej Czerwinski, Noreen A. McMahon, Joseph C. Eggleston,t Michael R. Boyd
(16) CHESON BD, BENNETT JM, RAI KR, ET AL:
Guidelines for clinical protocols for chronic lymphocytic leukemia: Recommendations of the National Cancer Institute-sponsored working group. Am J Hematol 29:152-163, 1988 (17) RAI KR, SAWTTSKY A, CRONKITE EP, ET AL;
Clinical staging of chronic lymphocytic leukemia. Blood 46:219-234, 1975 (18) BARLOGIE B, SMITH L, ALEXANIAN R: Effective
treatment of advanced multiple myeloma refractory to alkylating agents. N Engl J Med 310:1353-1356, 1984 (19) GREVER MR, LEIBY JM, KRAUT EH, ET AU
Low-dose deoxycoformycin in lymphoid malignancy. J Clin Oncol 3:1196-1201, 1985 (20) DILLMAN RO, MICK R, MCINTYRE OR: Pen-
tostatin in chronic lymphocytic leukemia: A phase II trial of cancer and leukemia group B. J Clin Oncol 4:433-438, 1989 (2/) CARSON DA, WASSON DB, TAETLE R, ET AU
Specific toxicify of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes. Blood 62:737-743, 1983
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4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [I4C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [I4C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per
30 minutes (mean ± SE = 547 ± 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean ± SE = 549 ± 60). In lung cancer tissue, values ranged from 0 to 2,566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean ± SE = 547 ± 60, P >.3). When patients were divided into smokers and current nonsmokers (no tobacco products smoked for > 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P > . l in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10;P>.30).The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted. [J Natl Cancer Inst 82:1420-1426,1990]
Received April 3, 1990; revised June 4, 1990; accepted June 14, 1990. T. L. McLemore, C. L. Litterst, B. P. Coudert, W. C. Hubbard, S. Adelberg, M. Czerwinski, M. R. Boyd, Program Development Research Group, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Md. M. C. Liu, N. A. McMahon, J. C. Eggleston, Departments of Medicine (Pulmonary Division) and Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Md. We thank Drs. A. Gazdar, J. Minna, 0 . Fodstad, G. D. Sorensen, and O.S. Pettengill for providing seed stock for lung cancer cell lines. We also thank Ms. Kathy Gill for assistance in the organization and processing of this manuscript. *Correspondence to: Theodore L. McLemore, M.D., 170 Eighth St. S.E.,Ste.C, Paris, TX 75460. tDeceased.
Journal of the National Cancer Institute
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Multiple agent chemotherapy (POACH) in previously treated and untreated patients with chronic lymphocytic leukemia. Leukemia 2: 157-164,1988 (9) CHESON BD: Current approaches to the chemotherapy of B-cell chronic lymphocytic leukemia: A review. Am J Hematol 32:72-77, 1989
(22) GREVER MR. SIAW MFE, JACOB WF, ET AL:
Materials and Methods Cell Lines Cell lines were provided from cryopreserved working seed stock or from viable cultures obtained from the In Vitro Cell Line Screening Project of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, Md). The cell lines have been previously characterized (25). Table 1 shows the histological Vol. 82, No. 17, September 5, 1990
TaWe 1. Histologic classification of established cell lines derived from human lung carcinomas Histology of cell lines Non-small cell lung cancer Adenocarcinoma Calu-6
Institution (source)*
Memorial Sloan-Kettering Cancer Center (New York, NY)
(ATCC)t A-549 NCI-H522 EKVX HOP-62 HOP-19
National Cancer Institute (Tumor Bank) National Cancer Institute (A. F. Gazdar) Norsk Hydro's Institute, Oslo, Norway ( 0 . Fodstad) National Cancer Institute} National Cancer Instituted
Adenosquamous carcinoma NCI-H125
National Cancer Institute (A. F. Gazdar)
Squamous cell carcinoma NCI-H520
National Cancer Institute (A. F. Gazdar)
Bronchioloalveolar carcinoma NCI-H322 NCI-H358
National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar)
Large cell undifferentiated lung carcinoma NCI-H460 HOP-18
National Cancer Institute (A. F. Gazdar) National Cancer Institute}
Small cell lung carcinoma NCI-H69 NCI-H146 NCI-H524 NCI-H82 DMS 114
National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) Dartmouth Medical School (Hanover, NH) (O. S. Pettengill)
Mixed small cell/large cell undifferentiated lung carcinoma HOP-27
National Cancer Institute}
•Characterization and origin of cell lines was previously summarized by Alley et al. (23) except where indicated. t ATCC = American Type Culture Collection, Rockville, Md. t Newly developed, unpublished lung cancer cell lines from our laboratories (42)
type and origin of each cell line used in this study. Cells were maintained in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum (Sterile System, HycloneR; Logan, Utah) and 2 mW Lglutamine at 37 °C in 5% CO2 and 95% air. RPMI-1640 cell culture medium, L-glutamine, and phosphate-buffered saline were obtained from Quality Biological, Inc., (Gaithersburg, Md). Trypsin-ethylene diaminetetra-acetic acid (1:250) was purchased from GIBCO Laboratories (Grand Island, NY). All cell lines grew as monolayers with the exception of NCIH69, which grew as floating aggregates, and HOP-27, which grew as a mixture of floating aggregates as well as colonies of attached cells. Polylysine (Sigma Chemical Co., St. Louis, Mo) was used to grow NCI-H524, NCI-H82, and NCI-H146 cell lines according to the protocol of Shipley and Ham (24). Cells were subcultured at a density of 0.5-1.5 x lO^cm2 and were used in the experiments upon reaching a
confluence of 80%~90%. Cell viability was assayed with the trypan blue dye exclusion technique, and only cell populations with viability greater than 80% were used in experiments. Acquisition and Preparation of Normal Lung Tissue and Lung Tumor Tissue Normal lung tissue and lung tumor tissues were obtained at the time of thoracotomy from 56 patients with lung cancer at the Johns Hopkins University Hospital (Baltimore, Md) and the Francis Scott Key Hospital (Baltimore, Md). The histological classification and distribution of the primary lung tumors included 24 squamous cell carcinomas, 15 adenocarcinomas, eight large cell undifferentiated carcinomas, four small cell carcinomas, three mixed cell carcinomas, and two bronchioloalveolar cell carcinomas. Other pertinent clinical information, including cigarette-smoking histories, is provided in table 2. After excision, both normal lung REPORTS 1421
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4-Ipomeanol (IPO) is the major component responsible for a fatal respiratory distress syndrome in cattle that results from ingestion of moldy sweet potatoes (L2). In experimental rodent models, pulmonary toxicity occurs through a cytochrome P450-dependent oxidation, with the formation of a highly reactive, electrophilic product that binds covalently to macromolecules within the pulmonary tissues (3-17). IPO preferentially causes necrosis to Clara cells of the respiratory tract (12,13,17) and, especially at higher doses, also causes damage to epithelial and endothelial cells, pulmonary edema, and hemorrhage (18,19). IPO has a propensity for selective bioactivation and cytotoxicity to lung cells, and this activity might also extend to lung tumor cells. For these reasons, the National Cancer Institute (NCI) (Bethesda, Md) is currently testing IPO as a potential anticancer agent for the treatment of human lung cancer (20). A previous study (27) demonstrated that two human bronchioloalveolar carcinoma cell lines, NCIH322 and NCI-H358, were capable of metabolizing IPO. Moreover, IPO treatment produced a substantial reduction in the size and number of lung tumors after intrabronchial implantation and propagation of one of these bronchioloalveolar cell carcinoma cell lines in afhymic nude mice (22). Because it was important to investigate a broader sampling of lung cancer cell lines and to compare the ability of fresh, normal human lung tissue or lung cancer tissue to activate IPO, we report here a study of IPO metabolism in 18 established cell lines derived from human lung cancer. Further, we extend this study to normal human lung tissue and lung cancer tissue obtained at thoracotomy from 56 patients with lung cancer.
Table 2. Smoking status of patients with lung cancer* Smoking status Current Nonsmokerst (19 patients) Age Previous smoking history}: Time since smoking cessation§ Smokers (37 patients) Age Smoking historyt
Mean No. of yr ± SE 64 ±2.3 56 ± 10.0 2.4 ± 0.7 60 ± 1.60 59 ± 4.4
•Twenty-eight women and 28 men were entered into the study. t Current nonsmokers are defined as those patients who have not smoked tobacco products for >6 months or who had never smoked. t Smoking history is expressed in pack-yrs. One pack-yr = 1 pack of cigarettes smoked per day for 1 yr. §For former smokers, the range of time since smoking cessation was 0.5-20 yr.
IPO Covalent Binding Assay The IPO covalent binding assay used in this study employs intact, viable cells and is a modification of methods previously reported (4,17). IPO and [14C]IPO in 95% ethanol were provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI. For assays involving lung cancer cell lines, tumor cell cultures were first washed with phosphatebuffered saline, trypsinized, centrifuged at 800 g for 5 minutes at 40 °C, resuspended in cold Krebs bicarbonate buffer (pH 7.4), and supplemented with 20 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid; cells were then counted. The incubate, containing 3-4 x 106 intact, viable cells in 2 mL of buffer, was then transferred to iced incubation vessels (chloride vials). For assays involving fresh tissues, intact, viable normal lung tissue or lung cancer tissue (8-10 fragments per vial) was transferred to the iced incubation vessels containing the incubation buffer as described above. The reaction was then started by the addition of 20 p-L of IPO (stock solution, 10 mg/mL in 0.9 sodium chloride) and 10 jiL of [ 14 C]IP0 (specific activity 27 mCi/mmol; radiochemical purity >98%) per tube (final IPO concentration, 0.67 mM). Vials containing cell lines or tissues were initially swirled to facilitate 1422
complete mixing and solubilization of the substrate, placed in a shaking water bath, and allowed to incubate for 30 minutes at 37 °C. Control vials were simultaneously processed and handled in exactly the same way, except that they were returned to the ice bath and boiled or treated with 1.0 mM piperonyl butoxide after mixing. Triplicate sets of control and experimental vials were then allowed to incubate for 30 minutes, and the reaction was terminated by addition of 2 mL of 20% trichloroacetic acid. The cell lines or tissue fragments were then immediately homogenized in a glass homogenizer with three strokes of a Teflon-coated pestle. The acid insoluble protein pellet was recovered by centrifugation at 1,500 g for 10 minutes and extracted ten times with 3-mL volumes of warm methanol (60 °C). After the final methanol wash, the protein residue was dissolved in 1.0 N sodium hydroxide by heating at 80 °C for 20 minutes. The final sodium hydroxide digest was used to determine radioactivity as well as total protein content. Radioactivity was determined in a scintillation counter (model LS 8201, Beckman Instruments, Fullerton, Calif) after solubilizing with Aquasol A and a standard scintillation cocktail mixture. The solution was neutralized by the addition of a small amount of glacial acetic acid prior to determination of radioisotope content. Protein content was determined by the method of Lowry et al. (26'); bovine serum albumin was used as a standard. Covalent binding of [14C]IPO was expressed as picomoles of IPO bound per milligram of protein per 30 min. After we corrected for the specific activ-
Results When the 18 lung cancer cell lines were tested for their ability to metabolize IPO, a wide variation in IPO metabolism was noted, with values of 248-1,047 pmol per milligram of protein per 30 minutes (overall mean ± SE = 547 ± 62.2) (fig. 1). The highest IPO covalent binding activities were demonstrated by an adenocarcinoma cell line (H522), a bronchioloalveolar cell line (H322), a large cell undifferentiated carcinoma (Hop 18), and a small cell carcinoma (DMS 114). When the cell lines were classified according to histological cell type, a wide variation in IPO binding was apparent within each cell type. However, all types demonstrated appreciable capacity to metabolize IPO (fig- 1). The metabolic activation of IPO was also evaluated in normal lung tissue and primary pulmonary carcinoma obtained at the time of thoracotomy from 56 patients with lung cancer. Similar mean levels (± SE) of IPO binding were noted in Journal of the National Cancer Institute
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tissue and lung tumor tissue were immediately placed on ice at 4 °C in Hanks' balanced salt solution. Samples were then minced on site into 1-mm3 fragments following removal of pleura and dissection and removal of tissue with gross abnormalities.
ity of the substrate, control experiments indicated that IPO covalent binding was linear for lung cancer cell lines or fresh tissue through 0.5 mM concentration of IPO and a 30-minute incubation period (six patients; data not shown). The effect of culture conditions on IPO activation in 18 lung cancer cell lines or fresh human lung tissue and lung cancer tissue were also investigated. Cell lines or fresh tissues were incubated on ice for 30 minutes, boiled, and treated with 1 mM piperonyl butoxide (a known P450 inhibitor) or incubated at 37 °C in a shaking water bath after addition of [I4C]IPO. Both icing and boiling strikingly decreased IPO binding in cell lines, normal lung tissue, or lung tumor tissue. Addition of piperonyl butoxide greatly decreased, but did not completely inhibit, IPO binding (five patients; P < . 0 1 , compared with controls; data not shown). These findings are similar to those previously reported in rodent pulmonary tissues (4,17,25) and human lung cancer cell lines (21). Therefore, for convenience, iced control samples were used for all cell line and fresh tissue experiments. For each experiment, the binding value for the iced control sample was subtracted from the experimental binding value to obtain the final IPO covalent binding value.
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.30). normal lung tissue (549 ± 60 pmol per mg of protein per 30 min: range, 12-2,007) and pulmonary carcinomas (547 ± 60 pmol per mg of protein per 30 min: range, 0-2,566) (P >.30, nonpaired, rwo-tailed Student's /-test; data not shown). When IPO metabolism was investigated in normal lung tissue and tumor tissue according to patient smoking history, no differences
Figure 2. 4-Ipomeanol metabolism in primary pulmonary carcinomas obtained at thoracotomy from 56 patients: analysis according to histological cell type. Columns = mean values; bars = SE. None of the differences shown by comparisons were statistically significant (P > .10 in all cases).
were noted between smokers and current nonsmokers (patients who had not smoked tobacco products >6 mo) in either tissue (P > . 1 in all instances; data not shown). In addition, no differences were noted for IPO metabolism in either male or female patients when normal lung tissue or tumor tissue was studied (P >.2 in all instances; data not shown).
Additionally, we divided the patient population into three groups to further study the relationship between IPO covalent binding in lung and tumor tissue from individual patients (fig. 4). Group I contained patients with higher IPO covalent binding in lung cancer tissue than in normal lung tissue; group II contained patients with similar IPO metabolism in normal lung tissue and lung tumor tissue; and group III contained patients with higher IPO binding in normal lung tissue than in the corresponding lung cancer tissue. Interestingly, approximately 50% of the patients demonstrated a strong positive correlation between IPO covalent binding in normal lung tissue and lung tumor tissue (r = .94; P £ o
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4-lpomeanol Normal Lung Tissue Binding (pmole/mg protein/30 min) Figure 4. Comparison of 4-ipomeanol covalent binding in normal lung tissue and tumor tissue from 56 patients with lung cancer. Each point represents the mean of triplicate determinations. Solid line represents a perfect positive linear correlation. Dashed lines represent the 95% confidence intervals calculated from multiple determinations of the correlation between IPO covalent binding in different normal lung tissue fragments from 20 patients with lung cancer. The patients were further subdivided into groups I, II, and III (as shown), with group II representing only those values within the confines of the dashed lines that indicate the 95% confidence intervals.
1424
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Figure 3. Frequency distribution of 4-ipomeanol covalent binding in normal lung tissue (A) and lung cancer tissue (B) from 56 patients with primary lung cancer. Values represent the mean of triplicate determinations.
with at least a 50-fold variation in metabolism of the compound. This range of activity is comparable with previously published data regarding metabolism of other substrates, such as benzo[a]pyrene, by selected P450 enzyme systems in human tissue (27-33). Interestingly, the P450 enzyme activity responsible for the metabolic activation of IPO in human lung tissue is not altered by cigarette smoking, as demonstrated by the data presented here. These results are unlike those for the previously studied P4501A1 system, which is present in human lung tissue and which is inducible by polycyclic hydrocarbon inducers, including those present in cigarette smoke condensate (27-33). The P450 system responsible for IPO metabolism appears to be a constitutively expressed enzyme system, but further studies are required for detailed classification of the specific human P450 enzyme involved in IPO activation.
This study extends previous investigations on a limited selection of cell lines (21) to include a diverse selection of lung cancer lines and fresh human lung cancer cell types. IPO activation and binding occurred to some extent in all of the cell lines tested. This result provides additional encouragement for testing IPO in human patients with lung cancer, particularly since all the common lung cancer cell types possess the ability to metabolize this agent. These observations differ slightly from those in a previous report (21) that found no detectable IPO covalent binding in two human small cell carcinoma cell lines. One of these lines (H69) was also evaluated in this study and was found to have a small amount of IPO-metabolizing activity. It should be emphasized, however, that this previous report (21) presented preliminary results of a study that used microsomal preparations derived from lung cancer cell lines incubated for only 10 minutes with [I4C]IPO. In our study, IPO metabolism was instead evaluated in viable, intact cell populations, and IPO binding was determined after incubation with radiolabeled IPO for 30 minutes. Furthermore, according to the assay conditions used in this study, IPO metabolism was linear through this 30-minute time frame. The assay for intact cells appears to provide a more physiological assay for measurement of IPO covalent binding. It may also more closely reflect the clinical situaJournal of the National Cancer Institute
The observation of altered regulation of the P450 enzyme system responsible for IPO metabolism in matched normal lung tissue and tumor tissue from individual patients is intriguing. Future studies to more precisely investigate this apparent abnormality in the regulation of the P450 isoenzyme pathways specific for metabolism of this agent might provide further elucidation of the mechanisms involved in this phenomenon. They might also provide additional insights for the developmental therapeutics of human lung cancer. The results of this study further support the rationale for clinical investigation of IPO in human lung cancer. In conclusion: 1) IPO metabolism was observed in the majority of normal lung tissues studied from 56 patients with lung Vol. 82, No. 17, September 5, 1990
cancer. 2) The metabolic activation of IPO was also observed in the majority of lung cancer tissues as well as the established lung cancer cell lines which were studied in the present report. 3) Altered regulation of IPO metabolism was observed in selected lung cancers from individual patients. 4) These abnormally regulated pathways might be useful in both the characterization of and development of treatment for human lung cancer.
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REPORTS 1425
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tion because, in the present report, fresh small cell carcinoma tissue fragments as well as small cell carcinoma cell lines demonstrated detectable metabolism of IPO. These results contrast with those of previous investigations showing a marked diminution in P450 enzyme activity during progression from preneoplastic to frankly neoplastic carcinomas of the liver in rodent models (34). Our results demonstrate appreciable IPO-metabolizing activity in all lung cancer cell lines and most fresh tumors studied. It is conceivable that major organ-site specific differences as well as species differences exist in P450 enzyme activity in tumors. Moreover, the extrapolation of results from animal systems to humans may not be straightforward for the P450 systems. Numerous biochemical and molecular genetic pathways are altered in human lung cancer (32,33,35-41), and these abnormal pathways might be useful in both the characterization and treatment of specific histological cell types for human lung cancer. The present data demonstrate that approximately 50% of human lung tumors express a sharply altered capacity for IPO metabolism compared with normal lung tissue from the same patient, which was obtained and analyzed simultaneously. These data suggest that regulation of the P450 enzyme system responsible for IPO metabolism is altered in these human pulmonary carcinomas. Previous studies have demonstrated altered regulation of another P450 gene in human pulmonary carcinomas: P4501A1, which is responsible for benzo[a]pyrene metabolism (32,33).
eds). New York: Plenum Press, 1983, pp 179197 (29) KOURI RE, LEVTNE AS, EDWARDS BK, ET AL:
Source of interindividual variations in aryl hydrocarbon hydroxylase in mitogen-activated human lymphocytes. In Banbury Report 16: Genetic Variability in Responses to Chemical Exposure. (Omewn G, Gelboin H, eds). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1984, pp 131-144 (30) KOURI RE, MCLEMORE T, JAISWAL AK, ET AL;
Current cellular assays for measuring clinical drug metabolizing capacity—impact of new molecular biologic techniques. In Ethnic Differences in Reactions to Drugs and Xenobiotics (Kalow W, Goeddle HW, Agarwal DP, eds). New York: Alan R. Liss, 1986, pp 453-469 (31) MCLEMORE TL, MARTIN RR: Chapter 1. Pul-
monary carcinogensis: Aryl hydrocarbon hydroxylase. In Cancer Treatment and Research, (Livingston RB, ed), vol 1, Lung Cancer 1. The Hague: Martinus-Nijhoff, 1981, pp 1-34 (32) MCLEMORE TL, ADELBERG S, LIU MC, ET AL:
Cytochrome P450IA1 gene expression in lung cancer patients: Evidence for cigarette smokeinduced expression in normal lung and altered gene regulation in primary pulmonary carcinomas. Cancer Res. In press (33) MCLEMORE TL, ADELBERG S, CZERWINSKI M,
ET AU Altered regulation of the cytochrome P450IA1 gene: Novel inducer-independent gene expression in pulmonary carcinoma cell lines. J Natl Cancer Inst 81:1787-1794,1989 (34) FARBER E: Cellular biochemistry of the stepwise development of cancer with chemicals: G.H.A. Clowes memorial lecture. Cancer Res 44:5463-5474, 1984
(38) GAZDAR AF, CARNEY DN, MINNA JD: The
biology of non-small cell lung cancer. Semin Oncol 10:3-19, 1983 (39) RODENHUIS S , VAN DE W E T E R D W M L , MOOI WJ, ET AL: Mutational activation of the K-ras oncogene: A possible pathogenic factor in adenocarcinoma of die lung. N Engl J Med 317:929-935, 1987 (40) FRIEND SH, DRYJA TP, WHNBERG RA: Onco-
genes and tumor-suppressing genes. N Engl J Med 318:618-622, 1988 (41) HUBBARD WC, ALLEY MC, MCLEMORE TL,
ET AL; Evidence for thromboxane biosynthesis in established cell lines derived from human lung carcinomas. Cancer Res 48:2674-2677, 1988
(35) MINNA JD, IHDE DC, GLATSTHN EJ: Lung
cancer Scalpels, beams, drugs, and probes. N EnglJMed 315:1411-1413, 1986 (36) YESNERR: Spectrum of lung cancer and ectopic hoimoncs. Pathol Annu 13:217-240, 1978
(42) MCLEMORE T, ALLEY M, LIU M, ET AL: Histo-
(37) MCLEMORE TL, HUBBARD WC, LITTERST C,
ET AL: Profiles of prostaglandin biosynthesis in normal lung and tumor tissue from lung cancer patients. Cancer Res 48:3140-3147, 1988
pathologic, biochemical, and molecular genetic characterization of four newly established pulmonary carcinoma cell lines. Proc Am Assoc Cancer Res 30:225, 1989
When you need cancer treatment information . . .
PDQ
• full-text prognostic and treatment options written for patients and other laypersons • • state-of-the-art statements, including detailed prognosis, staging, cellular classification, treatment overview, and response rates for nearly 80 different cancer diagnoses • more than 1,300 active clinical trials and 33 standard treatment protocols • names, addresses, and medical affiliations of more than 14,000 physicians devoted to the care of cancer patients • over 1,600 organizations and hospital centers that specialize in cancer research and treatment Complementing PDQ, NCI's CANCERLIT database is your best bet for comprehensive coverage of current and historic cancer research and therapy. Searched like MEDLINE, CANCERLIT is a bibliographic database designed to bring you worldwide information sources in the fields of cancer diagnosis and therapy, carcinogenesis, virology, and biology. Updated monthly, CANCERLIT includes: • all aspects of cancer research and clinical therapy • more than 600,000 citations, most with abstracts • meeting papers, books, U.S. Government reports, and dissertations, as well as published journal articles
i( For more information, contact: NCI/ICIC, BIdg. 82, Rm. 119, Bethesda, MD 20892, 301496-7403
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sncxMAi. ITUTI
Journal of the National Cancer Institute
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PDQ is your database of choice. Updated monthly, PDQ is the National Cancer Institute's (NCI) primary online resource for the latest and most comprehensive cancer treatment information available, including:
Treatment of chronic lymphocytic leukemia using chlorambucil and prednisone with or without cycle-active consolidation chemotherapy. A Southeastern Cancer Study Group trial. Cancer 58:1185-1192, 1986 (5) BINET JL, LEPORRIER M, DIGHIERO G, ET AL: A
clinical staging system for chronic lymphocytic leukemia: Prognostic significance. Cancer 40:855-864, 1977 (6) LIEPMAN M, VOTAW ML: The treatment of
chronic lymphocytic leukemia with COP chemotherapy. Cancer 41:1664-1669, 1978 (7) KEMPIN S, LEE B r III, THALER HT, ET AL:
Combination chemotherapy of advanced chronic lymphocytic leukemia: The M-2 protocol (vincristine, BCNU, cyclophosphamide, melphalan, and prednisone). Blood 60:11101121, 1982 (8) KEATING Ml, SCOUROS M, MURPHY S, ET AL:
The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy. Blood 57:406-417, 1981 (23) Ho AD, GANESHAGURU K, KNAUF WU, ET AL:
Clinical response to deoxycoformycin in chronic lymphoid neoplasms and biochemical changes in circulating malignant cells in vivo. Blood 6:1884-1890, 1988 (24) PIRO LD, CARRERA CJ, BEUTLER E, ET AL:
2'-Chlorodeoxyadenosine: An effective new agent for the treatment of chronic lymphocytic leukemia. Blood 72:1069-1073, 1988 (25) GREVER MR, KOPECKY KJ, COLTMAN CA,
ET AL: Fludarabine monophosphate: A potentially useful agent in chronic lymphocytic leukemia. Nouv Rev Fr Hematol 30:457-459, 1988 (26) KEATING MJ, KANTARJIAN H.TALPAZM, ETAL:
Fludarabine: A new agent with major activity against chronic lymphocytic leukemia. Blood 74:19-25,1989 (27) O'DWYER PJ, SPIERS ASD, MARSONI S: Asso-
ciation of severe and fatal infections and treatment with pentostatin. Cancer Treat 70: 1117-1120, 1986
(10) FRENCH COOPERATIVE GROUP ON CHRONIC LYM-
PHOCYTIC LEUKAEMIA: Long-term results of the
CHOP regimen in stage C chronic lymphocytic leukaemia. Br J Haematol 73:334-340, 1989 (//) O'DWYER PJ, WAGNER B, LEYLAND-JONES B,
ET AL: 2'-Deoxycoformycin (pentostatin) for lymphoid malignancies. Rational development of an active new drug. Ann Intern Med 108:733-743, 1988 (12) KRAUT EH, BOURONCLE BA, GREVER MR:
Low-dose deoxycoformycin in the treatment of hairy cell leukemia. Blood 68:1119-1122, 1986 (13) SPIERS ASD, MOORE D, CASSILETH PA, ET AL:
Remissions in hairy-cell leukemia with pentostatin (2'-deoxycoformycin). N Engl J Med 316:825-830, 1987 (14) Ho AD, THALER J, MANDELLJ F, ET AL: Re-
sponse to pentostatin in hairy-cell leukemia refractory to interferon-alpha. The European Organization for Research and Treatment of Cancer Leukemia Cooperative Group. J Clin Oncol 10:1533-1538, 1989 (15) MILLER AB, HOOGSTRATEN B, STAQUET M,
ET AL: Reporting results of cancer treatment. Cancer47:207-214, 1981
Metabolic Activation of 4-Ipomeanol in Human Lung, Primary Pulmonary Carcinomas, and Established Human Pulmonary Carcinoma Cell Lines Theodore L. McLemore* Charles L. Litterst, Bruno P. Coudert, Mark C. Liu, Walter C. Hubbard, Steven Adelberg, Maciej Czerwinski, Noreen A. McMahon, Joseph C. Eggleston,t Michael R. Boyd
(16) CHESON BD, BENNETT JM, RAI KR, ET AL:
Guidelines for clinical protocols for chronic lymphocytic leukemia: Recommendations of the National Cancer Institute-sponsored working group. Am J Hematol 29:152-163, 1988 (17) RAI KR, SAWTTSKY A, CRONKITE EP, ET AL;
Clinical staging of chronic lymphocytic leukemia. Blood 46:219-234, 1975 (18) BARLOGIE B, SMITH L, ALEXANIAN R: Effective
treatment of advanced multiple myeloma refractory to alkylating agents. N Engl J Med 310:1353-1356, 1984 (19) GREVER MR, LEIBY JM, KRAUT EH, ET AU
Low-dose deoxycoformycin in lymphoid malignancy. J Clin Oncol 3:1196-1201, 1985 (20) DILLMAN RO, MICK R, MCINTYRE OR: Pen-
tostatin in chronic lymphocytic leukemia: A phase II trial of cancer and leukemia group B. J Clin Oncol 4:433-438, 1989 (2/) CARSON DA, WASSON DB, TAETLE R, ET AU
Specific toxicify of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes. Blood 62:737-743, 1983
1420
4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [I4C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [I4C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per
30 minutes (mean ± SE = 547 ± 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean ± SE = 549 ± 60). In lung cancer tissue, values ranged from 0 to 2,566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean ± SE = 547 ± 60, P >.3). When patients were divided into smokers and current nonsmokers (no tobacco products smoked for > 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P > . l in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10;P>.30).The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted. [J Natl Cancer Inst 82:1420-1426,1990]
Received April 3, 1990; revised June 4, 1990; accepted June 14, 1990. T. L. McLemore, C. L. Litterst, B. P. Coudert, W. C. Hubbard, S. Adelberg, M. Czerwinski, M. R. Boyd, Program Development Research Group, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Md. M. C. Liu, N. A. McMahon, J. C. Eggleston, Departments of Medicine (Pulmonary Division) and Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Md. We thank Drs. A. Gazdar, J. Minna, 0 . Fodstad, G. D. Sorensen, and O.S. Pettengill for providing seed stock for lung cancer cell lines. We also thank Ms. Kathy Gill for assistance in the organization and processing of this manuscript. *Correspondence to: Theodore L. McLemore, M.D., 170 Eighth St. S.E.,Ste.C, Paris, TX 75460. tDeceased.
Journal of the National Cancer Institute
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Multiple agent chemotherapy (POACH) in previously treated and untreated patients with chronic lymphocytic leukemia. Leukemia 2: 157-164,1988 (9) CHESON BD: Current approaches to the chemotherapy of B-cell chronic lymphocytic leukemia: A review. Am J Hematol 32:72-77, 1989
(22) GREVER MR. SIAW MFE, JACOB WF, ET AL:
Materials and Methods Cell Lines Cell lines were provided from cryopreserved working seed stock or from viable cultures obtained from the In Vitro Cell Line Screening Project of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, Md). The cell lines have been previously characterized (25). Table 1 shows the histological Vol. 82, No. 17, September 5, 1990
TaWe 1. Histologic classification of established cell lines derived from human lung carcinomas Histology of cell lines Non-small cell lung cancer Adenocarcinoma Calu-6
Institution (source)*
Memorial Sloan-Kettering Cancer Center (New York, NY)
(ATCC)t A-549 NCI-H522 EKVX HOP-62 HOP-19
National Cancer Institute (Tumor Bank) National Cancer Institute (A. F. Gazdar) Norsk Hydro's Institute, Oslo, Norway ( 0 . Fodstad) National Cancer Institute} National Cancer Instituted
Adenosquamous carcinoma NCI-H125
National Cancer Institute (A. F. Gazdar)
Squamous cell carcinoma NCI-H520
National Cancer Institute (A. F. Gazdar)
Bronchioloalveolar carcinoma NCI-H322 NCI-H358
National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar)
Large cell undifferentiated lung carcinoma NCI-H460 HOP-18
National Cancer Institute (A. F. Gazdar) National Cancer Institute}
Small cell lung carcinoma NCI-H69 NCI-H146 NCI-H524 NCI-H82 DMS 114
National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) National Cancer Institute (A. F. Gazdar) Dartmouth Medical School (Hanover, NH) (O. S. Pettengill)
Mixed small cell/large cell undifferentiated lung carcinoma HOP-27
National Cancer Institute}
•Characterization and origin of cell lines was previously summarized by Alley et al. (23) except where indicated. t ATCC = American Type Culture Collection, Rockville, Md. t Newly developed, unpublished lung cancer cell lines from our laboratories (42)
type and origin of each cell line used in this study. Cells were maintained in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum (Sterile System, HycloneR; Logan, Utah) and 2 mW Lglutamine at 37 °C in 5% CO2 and 95% air. RPMI-1640 cell culture medium, L-glutamine, and phosphate-buffered saline were obtained from Quality Biological, Inc., (Gaithersburg, Md). Trypsin-ethylene diaminetetra-acetic acid (1:250) was purchased from GIBCO Laboratories (Grand Island, NY). All cell lines grew as monolayers with the exception of NCIH69, which grew as floating aggregates, and HOP-27, which grew as a mixture of floating aggregates as well as colonies of attached cells. Polylysine (Sigma Chemical Co., St. Louis, Mo) was used to grow NCI-H524, NCI-H82, and NCI-H146 cell lines according to the protocol of Shipley and Ham (24). Cells were subcultured at a density of 0.5-1.5 x lO^cm2 and were used in the experiments upon reaching a
confluence of 80%~90%. Cell viability was assayed with the trypan blue dye exclusion technique, and only cell populations with viability greater than 80% were used in experiments. Acquisition and Preparation of Normal Lung Tissue and Lung Tumor Tissue Normal lung tissue and lung tumor tissues were obtained at the time of thoracotomy from 56 patients with lung cancer at the Johns Hopkins University Hospital (Baltimore, Md) and the Francis Scott Key Hospital (Baltimore, Md). The histological classification and distribution of the primary lung tumors included 24 squamous cell carcinomas, 15 adenocarcinomas, eight large cell undifferentiated carcinomas, four small cell carcinomas, three mixed cell carcinomas, and two bronchioloalveolar cell carcinomas. Other pertinent clinical information, including cigarette-smoking histories, is provided in table 2. After excision, both normal lung REPORTS 1421
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4-Ipomeanol (IPO) is the major component responsible for a fatal respiratory distress syndrome in cattle that results from ingestion of moldy sweet potatoes (L2). In experimental rodent models, pulmonary toxicity occurs through a cytochrome P450-dependent oxidation, with the formation of a highly reactive, electrophilic product that binds covalently to macromolecules within the pulmonary tissues (3-17). IPO preferentially causes necrosis to Clara cells of the respiratory tract (12,13,17) and, especially at higher doses, also causes damage to epithelial and endothelial cells, pulmonary edema, and hemorrhage (18,19). IPO has a propensity for selective bioactivation and cytotoxicity to lung cells, and this activity might also extend to lung tumor cells. For these reasons, the National Cancer Institute (NCI) (Bethesda, Md) is currently testing IPO as a potential anticancer agent for the treatment of human lung cancer (20). A previous study (27) demonstrated that two human bronchioloalveolar carcinoma cell lines, NCIH322 and NCI-H358, were capable of metabolizing IPO. Moreover, IPO treatment produced a substantial reduction in the size and number of lung tumors after intrabronchial implantation and propagation of one of these bronchioloalveolar cell carcinoma cell lines in afhymic nude mice (22). Because it was important to investigate a broader sampling of lung cancer cell lines and to compare the ability of fresh, normal human lung tissue or lung cancer tissue to activate IPO, we report here a study of IPO metabolism in 18 established cell lines derived from human lung cancer. Further, we extend this study to normal human lung tissue and lung cancer tissue obtained at thoracotomy from 56 patients with lung cancer.
Table 2. Smoking status of patients with lung cancer* Smoking status Current Nonsmokerst (19 patients) Age Previous smoking history}: Time since smoking cessation§ Smokers (37 patients) Age Smoking historyt
Mean No. of yr ± SE 64 ±2.3 56 ± 10.0 2.4 ± 0.7 60 ± 1.60 59 ± 4.4
•Twenty-eight women and 28 men were entered into the study. t Current nonsmokers are defined as those patients who have not smoked tobacco products for >6 months or who had never smoked. t Smoking history is expressed in pack-yrs. One pack-yr = 1 pack of cigarettes smoked per day for 1 yr. §For former smokers, the range of time since smoking cessation was 0.5-20 yr.
IPO Covalent Binding Assay The IPO covalent binding assay used in this study employs intact, viable cells and is a modification of methods previously reported (4,17). IPO and [14C]IPO in 95% ethanol were provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI. For assays involving lung cancer cell lines, tumor cell cultures were first washed with phosphatebuffered saline, trypsinized, centrifuged at 800 g for 5 minutes at 40 °C, resuspended in cold Krebs bicarbonate buffer (pH 7.4), and supplemented with 20 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid; cells were then counted. The incubate, containing 3-4 x 106 intact, viable cells in 2 mL of buffer, was then transferred to iced incubation vessels (chloride vials). For assays involving fresh tissues, intact, viable normal lung tissue or lung cancer tissue (8-10 fragments per vial) was transferred to the iced incubation vessels containing the incubation buffer as described above. The reaction was then started by the addition of 20 p-L of IPO (stock solution, 10 mg/mL in 0.9 sodium chloride) and 10 jiL of [ 14 C]IP0 (specific activity 27 mCi/mmol; radiochemical purity >98%) per tube (final IPO concentration, 0.67 mM). Vials containing cell lines or tissues were initially swirled to facilitate 1422
complete mixing and solubilization of the substrate, placed in a shaking water bath, and allowed to incubate for 30 minutes at 37 °C. Control vials were simultaneously processed and handled in exactly the same way, except that they were returned to the ice bath and boiled or treated with 1.0 mM piperonyl butoxide after mixing. Triplicate sets of control and experimental vials were then allowed to incubate for 30 minutes, and the reaction was terminated by addition of 2 mL of 20% trichloroacetic acid. The cell lines or tissue fragments were then immediately homogenized in a glass homogenizer with three strokes of a Teflon-coated pestle. The acid insoluble protein pellet was recovered by centrifugation at 1,500 g for 10 minutes and extracted ten times with 3-mL volumes of warm methanol (60 °C). After the final methanol wash, the protein residue was dissolved in 1.0 N sodium hydroxide by heating at 80 °C for 20 minutes. The final sodium hydroxide digest was used to determine radioactivity as well as total protein content. Radioactivity was determined in a scintillation counter (model LS 8201, Beckman Instruments, Fullerton, Calif) after solubilizing with Aquasol A and a standard scintillation cocktail mixture. The solution was neutralized by the addition of a small amount of glacial acetic acid prior to determination of radioisotope content. Protein content was determined by the method of Lowry et al. (26'); bovine serum albumin was used as a standard. Covalent binding of [14C]IPO was expressed as picomoles of IPO bound per milligram of protein per 30 min. After we corrected for the specific activ-
Results When the 18 lung cancer cell lines were tested for their ability to metabolize IPO, a wide variation in IPO metabolism was noted, with values of 248-1,047 pmol per milligram of protein per 30 minutes (overall mean ± SE = 547 ± 62.2) (fig. 1). The highest IPO covalent binding activities were demonstrated by an adenocarcinoma cell line (H522), a bronchioloalveolar cell line (H322), a large cell undifferentiated carcinoma (Hop 18), and a small cell carcinoma (DMS 114). When the cell lines were classified according to histological cell type, a wide variation in IPO binding was apparent within each cell type. However, all types demonstrated appreciable capacity to metabolize IPO (fig- 1). The metabolic activation of IPO was also evaluated in normal lung tissue and primary pulmonary carcinoma obtained at the time of thoracotomy from 56 patients with lung cancer. Similar mean levels (± SE) of IPO binding were noted in Journal of the National Cancer Institute
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tissue and lung tumor tissue were immediately placed on ice at 4 °C in Hanks' balanced salt solution. Samples were then minced on site into 1-mm3 fragments following removal of pleura and dissection and removal of tissue with gross abnormalities.
ity of the substrate, control experiments indicated that IPO covalent binding was linear for lung cancer cell lines or fresh tissue through 0.5 mM concentration of IPO and a 30-minute incubation period (six patients; data not shown). The effect of culture conditions on IPO activation in 18 lung cancer cell lines or fresh human lung tissue and lung cancer tissue were also investigated. Cell lines or fresh tissues were incubated on ice for 30 minutes, boiled, and treated with 1 mM piperonyl butoxide (a known P450 inhibitor) or incubated at 37 °C in a shaking water bath after addition of [I4C]IPO. Both icing and boiling strikingly decreased IPO binding in cell lines, normal lung tissue, or lung tumor tissue. Addition of piperonyl butoxide greatly decreased, but did not completely inhibit, IPO binding (five patients; P < . 0 1 , compared with controls; data not shown). These findings are similar to those previously reported in rodent pulmonary tissues (4,17,25) and human lung cancer cell lines (21). Therefore, for convenience, iced control samples were used for all cell line and fresh tissue experiments. For each experiment, the binding value for the iced control sample was subtracted from the experimental binding value to obtain the final IPO covalent binding value.
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.30). normal lung tissue (549 ± 60 pmol per mg of protein per 30 min: range, 12-2,007) and pulmonary carcinomas (547 ± 60 pmol per mg of protein per 30 min: range, 0-2,566) (P >.30, nonpaired, rwo-tailed Student's /-test; data not shown). When IPO metabolism was investigated in normal lung tissue and tumor tissue according to patient smoking history, no differences
Figure 2. 4-Ipomeanol metabolism in primary pulmonary carcinomas obtained at thoracotomy from 56 patients: analysis according to histological cell type. Columns = mean values; bars = SE. None of the differences shown by comparisons were statistically significant (P > .10 in all cases).
were noted between smokers and current nonsmokers (patients who had not smoked tobacco products >6 mo) in either tissue (P > . 1 in all instances; data not shown). In addition, no differences were noted for IPO metabolism in either male or female patients when normal lung tissue or tumor tissue was studied (P >.2 in all instances; data not shown).
Additionally, we divided the patient population into three groups to further study the relationship between IPO covalent binding in lung and tumor tissue from individual patients (fig. 4). Group I contained patients with higher IPO covalent binding in lung cancer tissue than in normal lung tissue; group II contained patients with similar IPO metabolism in normal lung tissue and lung tumor tissue; and group III contained patients with higher IPO binding in normal lung tissue than in the corresponding lung cancer tissue. Interestingly, approximately 50% of the patients demonstrated a strong positive correlation between IPO covalent binding in normal lung tissue and lung tumor tissue (r = .94; P £ o
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4-lpomeanol Normal Lung Tissue Binding (pmole/mg protein/30 min) Figure 4. Comparison of 4-ipomeanol covalent binding in normal lung tissue and tumor tissue from 56 patients with lung cancer. Each point represents the mean of triplicate determinations. Solid line represents a perfect positive linear correlation. Dashed lines represent the 95% confidence intervals calculated from multiple determinations of the correlation between IPO covalent binding in different normal lung tissue fragments from 20 patients with lung cancer. The patients were further subdivided into groups I, II, and III (as shown), with group II representing only those values within the confines of the dashed lines that indicate the 95% confidence intervals.
1424
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Figure 3. Frequency distribution of 4-ipomeanol covalent binding in normal lung tissue (A) and lung cancer tissue (B) from 56 patients with primary lung cancer. Values represent the mean of triplicate determinations.
with at least a 50-fold variation in metabolism of the compound. This range of activity is comparable with previously published data regarding metabolism of other substrates, such as benzo[a]pyrene, by selected P450 enzyme systems in human tissue (27-33). Interestingly, the P450 enzyme activity responsible for the metabolic activation of IPO in human lung tissue is not altered by cigarette smoking, as demonstrated by the data presented here. These results are unlike those for the previously studied P4501A1 system, which is present in human lung tissue and which is inducible by polycyclic hydrocarbon inducers, including those present in cigarette smoke condensate (27-33). The P450 system responsible for IPO metabolism appears to be a constitutively expressed enzyme system, but further studies are required for detailed classification of the specific human P450 enzyme involved in IPO activation.
This study extends previous investigations on a limited selection of cell lines (21) to include a diverse selection of lung cancer lines and fresh human lung cancer cell types. IPO activation and binding occurred to some extent in all of the cell lines tested. This result provides additional encouragement for testing IPO in human patients with lung cancer, particularly since all the common lung cancer cell types possess the ability to metabolize this agent. These observations differ slightly from those in a previous report (21) that found no detectable IPO covalent binding in two human small cell carcinoma cell lines. One of these lines (H69) was also evaluated in this study and was found to have a small amount of IPO-metabolizing activity. It should be emphasized, however, that this previous report (21) presented preliminary results of a study that used microsomal preparations derived from lung cancer cell lines incubated for only 10 minutes with [I4C]IPO. In our study, IPO metabolism was instead evaluated in viable, intact cell populations, and IPO binding was determined after incubation with radiolabeled IPO for 30 minutes. Furthermore, according to the assay conditions used in this study, IPO metabolism was linear through this 30-minute time frame. The assay for intact cells appears to provide a more physiological assay for measurement of IPO covalent binding. It may also more closely reflect the clinical situaJournal of the National Cancer Institute
The observation of altered regulation of the P450 enzyme system responsible for IPO metabolism in matched normal lung tissue and tumor tissue from individual patients is intriguing. Future studies to more precisely investigate this apparent abnormality in the regulation of the P450 isoenzyme pathways specific for metabolism of this agent might provide further elucidation of the mechanisms involved in this phenomenon. They might also provide additional insights for the developmental therapeutics of human lung cancer. The results of this study further support the rationale for clinical investigation of IPO in human lung cancer. In conclusion: 1) IPO metabolism was observed in the majority of normal lung tissues studied from 56 patients with lung Vol. 82, No. 17, September 5, 1990
cancer. 2) The metabolic activation of IPO was also observed in the majority of lung cancer tissues as well as the established lung cancer cell lines which were studied in the present report. 3) Altered regulation of IPO metabolism was observed in selected lung cancers from individual patients. 4) These abnormally regulated pathways might be useful in both the characterization of and development of treatment for human lung cancer.
References (7) BOYD MR, WILSON BJ: Isolation and character-
ization of 4-ipomeanol, a lung toxic furanoterpemoid produced by sweet potatoes (ipomoca batatas). J Agric Food Chem 20:428-430,1972 (2) BOYD MR, BURKA LT, HARRIS TM, ET AL:
Lung toxin furanoterpenoids produced by sweet potatoes (ipomoea batatas) following microbial infection. Biochem Biophys Acta 337:184— 195, 1974 (3) BOYD MR, BURKA LT, WILSON BJ: Distribu-
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tion because, in the present report, fresh small cell carcinoma tissue fragments as well as small cell carcinoma cell lines demonstrated detectable metabolism of IPO. These results contrast with those of previous investigations showing a marked diminution in P450 enzyme activity during progression from preneoplastic to frankly neoplastic carcinomas of the liver in rodent models (34). Our results demonstrate appreciable IPO-metabolizing activity in all lung cancer cell lines and most fresh tumors studied. It is conceivable that major organ-site specific differences as well as species differences exist in P450 enzyme activity in tumors. Moreover, the extrapolation of results from animal systems to humans may not be straightforward for the P450 systems. Numerous biochemical and molecular genetic pathways are altered in human lung cancer (32,33,35-41), and these abnormal pathways might be useful in both the characterization and treatment of specific histological cell types for human lung cancer. The present data demonstrate that approximately 50% of human lung tumors express a sharply altered capacity for IPO metabolism compared with normal lung tissue from the same patient, which was obtained and analyzed simultaneously. These data suggest that regulation of the P450 enzyme system responsible for IPO metabolism is altered in these human pulmonary carcinomas. Previous studies have demonstrated altered regulation of another P450 gene in human pulmonary carcinomas: P4501A1, which is responsible for benzo[a]pyrene metabolism (32,33).
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pathologic, biochemical, and molecular genetic characterization of four newly established pulmonary carcinoma cell lines. Proc Am Assoc Cancer Res 30:225, 1989
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