Mechanism of natural resistance to N6-(delta2-isopentenyl)adenosine in cultured cells.

Twenty-one cell lines (six human lines, and nine mouse lines) were compared with respect to inhibition of growth by N-6-(delta-2-isopentenyl)adenosine (IPAR). Six of these, mouse Sarcoma 180, Ehrilich ascites carcinoma, mammary adenocarconoma (TA3), leukemia L1210, mouse kidney, and canine kidney cells, differed by up to 16-fold with respect to their sensitivity and were chosen for further study. One factor contributing to the resistance was a slower formation of intracellular 5-monophosphate of IPAR (IPAMP) due to reduced adenosine kinase activity. Because of this slower formation of IPAMP in the resistant cells, a higher extracellular IPAR was required for the maintenance of equal intracellular IPAMP levels. Regardless of the degree of resistance, the rate of decay of intracellular IPAMP was similar and very rapid, with a half-life of 37 plus or minus 5 min. In the sensitive cells IPAMP was cleaved back to IPAR, while in the resistant cells IPAR was cleaved further to the free base, N-6-(delta-2-isopentenyl)adenine (IPA), which accumulated in the medium. The rate of formation of IPA constitutes an irreversible inactivation of IPAR, because IPA is not converted back to IPAMP and is not growth inhibitory. In one of the resistant cells (mouse kidney) the inactivation was so rapid that in 1 hr 25% of the extracellular (30 muM) IPAR was converted to IPA.

Modeling of the time-dependency of in vitro drug cytotoxicity and resistance.

For potential clinical extrapolation of in vitro findings, it is of interest to relate the measured effect of an anticancer agent to concentration and exposure time. The Hill model (A. V. Hill, J. Physiol., 40: iv-vii, 1910) is commonly used to describe pharmacodynamic (PD) effects, including drug-induced growth inhibition of cancer cells in vitro. The IC(X)n x T = k relationship, in which IC(X) is the concentration of agent required to reduce cell growth by X%, T is the exposure time, and n and k are estimable parameters, was first applied to bacterial disinfectant action and then was successfully used to model anticancer drug potency as a function of exposure time (D. J. Adams, Cancer Res., 49: 6615-6620, 1989). Our goal was to create a new global PD modeling paradigm to facilitate the quantitative assessment of the growth-inhibitory effect of anticancer agents as a function of concentration and exposure time. Wild-type human ovarian A2780 and ileocecal HCT-8 carcinoma cells and sublines that were resistant to cisplatin (A2780/CP3), doxorubicin (A2780/DX5B), and raltitrexed (RTX) (HCT-8/DW2) were exposed to various anticancer agents, cisplatin, doxorubicin, paclitaxel, trimetrexate, RTX, methotrexate, and AG2034, for periods ranging from 1 to 96 h. Cell growth inhibition was measured with the sulforhodamine B protein dye assay. Patterns of time-dependency of drug potency, slope of the concentration-effect curves, and relative degree of resistance were characterized. Empirical mathematical expressions were built into a global concentration-time-effect model. The global PD model was then fit to the concentration-time-effect data with iteratively reweighted nonlinear regression. Under specific treatment conditions, the examination of the slope and the shape of the concentration-effect curves revealed a large heterogeneity in drug response, e.g., shallow concentration-effect curve or double or triple Hill "roller coaster" concentration-effect curve. These patterns, which were observed at intermediate exposure times in parental and resistant cells for paclitaxel and trimetrexate or only in resistant HCT-8/DW2 cells for RTX, methotrexate, and AG2034, revealed mechanistic insights for the former cases but possible methodological artifacts for the latter cases. The comprehensive PD modeling of the cytotoxic effect of anticancer agents showed that it was possible to modulate drug effect, response heterogeneity, and drug resistance by altering the time of exposure to the agents. This approach will be useful for: (a) describing complex concentration-time-effect surfaces; (b) refining biological interpretations of data; (c) providing insights on mechanisms of drug action and resistance; and (d) generating leads for clinical use of anticancer drugs.

Colony growth in soft agar of human melanoma, sarcoma, and lung carcinoma cells disaggregated by mechanical and enzymatic methods.

The effect of mechanical and enzymatic disaggregation on human malignant melanoma, soft-tissue sarcoma and lung carcinoma colony growth in soft agar was studied. The enzymatic disaggregation was advantageous in most cases of melanoma and sarcoma, giving a larger number of colonies and increasing the probability of achieving growth in soft agar. Enzymatically treated pulmonary carcinoma cell populations had lower clonogeneic potential, especially in the case of anaplastic carcinomas. Morphological studies showed that the cells growing in soft-agar colonies had the same characteristics as those of the original tumor. A linear relationship was obtained between the number of enzymatically and mechanically treated tumor cells plated and the number of colonies. Delayed plating decreased the number of colonies.

Purification of soluble human melanoma-associated antigens.

Soluble extracts of melanoma specimens were prepared by 3 M KCl extraction. Delayed cutaneous hypersensitivity reactions to these antigens were noted in 25 of 39 melanoma patients and 7 of 30 patients with other neoplasms. Only 4 of 34 melanoma patients reacted to an extract of autologous muscle. Maximum reactivity to these antigens occurred at 24 hr and was demonstrated histologically by skin biopsy. Chromatography on Sephadex G-150 resulted in two fractions that elicited delayed cutaneous hypersensitivity reactions in melanoma patients. These fractions were subjected to polyacrylamide gel electrophoresis. The first region of the Sephadex 1 gel reacted in 8 of 13 melanoma patients and only 1 of 7 patients with other neoplasms. Some activity was found in other regions of the gel. Skin test activity was confined to the second polyacrylamide gel electrophoresis region of the Sephadex II gel, to which 7 of 9 melanoma patients reacted compared with 1 of 7 patients with other tumors. Recovery of antigenic activity in excess of the total present in the original extract after partial purification indicated that inhibitors of delayed cutaneous hypersensitivity reactions present in the crude KCl extract were removed. A 20-fold increase in antigenic activity per unit protein was achieved.

Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors.

Characterization of cells comprising solid tumors will facilitate the rational design of cancer chemotherapy for individual patients. We have prepared cell suspensions from human melanoma, sarcoma, and lung tumors by thinly slicing the tissue with a microtome and scalpels (mechanical release), followed by treatment with a mixture of collagenase II and DNase I (enzymatic release). This method of disaggregation resulted in two cell suspensions for each tumor specimen, and we characterized these suspensions by assessing their dye exclusion capability, ribonucleoside triphosphate pools, cytological profile and clonogenicity in soft agar. The enzymatic method thus yields cells in addition to those obtainable by a mild mechanical procedure, and these cells are similar in cytological profile and clonogenicity in soft agar to those released mechanically. Furthermore, the enzymatically released population is superior to that released mechanically for purposes requiring large numbers of dye-excluding cells having intact ribonucleotide pools.

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation.

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.

Growth of human urological tumors on extracellular matrix as a model for the in vitro cultivation of primary human tumor explants.

The goal of this study was to establish an optimal in vitro growth assay system for human urological tumor explants. Bovine corneal endotelial cell extracellular matrix (ECM) coated dishes were evaluated as a growth substrate for tumor cultures. Growth success for different urological carcinomas (prostatic, bladder, kidney, and testicular) was compared after seeding fresh surgical explants onto bovine corneal endothelial cell ECM and plastic culture flasks. Tumor samples were disaggregated enzymatically, and 1 X 10(4) cells were seeded onto the different substrates using RPMI 1640 medium containing 10% fetal calf serum and/or different growth factors, nutrients, and hormones. Cell growth on ECM was quantitated on days 7-15 by [3H]thymidine uptake, cell counting, and total protein. Tumor cells were characterized by flow cytometry and cytology. It was observed that ECM provides superior culture conditions for urological carcinomas. By increasing the initial number of cells plated on ECM and by adding different growth factors or hormones, the growth rate for specific tumor types was increased significantly. Several tumors (11 cases) grown on ECM were examined under the light microscope, and in all cases pre- and post-cytology confirmed malignancy. Tumor cells maintained on ECM and transplanted into nude mide retained their tumorigenic and morphological characteristics. Clinically aggressive tumors were associated with extensive ECM degradation. In addition, the growth of fresh human tumors on ECM provides a biologically relevant model system (for assessing the invasiveness of tumors in vitro) and should also be useful for drug evaluation studies.

P-glycoprotein in human sarcoma: evidence for multidrug resistance.

Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.

S-phase modulation by irinotecan: pilot studies in advanced solid tumors.

Two studies of irinotecan (CPT-11) followed 24 h later by an antimetabolite were conducted. The objectives of the studies were: (1) to determine whether the increase in S-phase in tumor cells seen 24 h after CPT-11 administration in animal studies is seen in advanced solid tumors in patients, (2) to determine the dose of CPT-11 required to produce this effect, (3) to compare two methods (immunohistochemistry, IHC, for cyclin A, and DNA flow cytometry, FC) for evaluating S-phase in tumor biopsies from patients, and (4) to establish the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of CPT-11, given 24 h before gemcitabine (GEM, 1000 mg/m(2)). In one study CPT-11 was followed 24 h later by 5-fluorouracil (5-FU), 400 mg/m(2) per week for 4 weeks every 6 weeks. Tumor biopsies were obtained before and 24 h after CPT-11 administration before administration of 5-FU and assayed for S-phase by IHC for cyclin A and by FC. The starting dose of CPT-11 was 80 mg/m(2) per week with subsequent exploration of 40 and 60 mg/m(2) per week to establish the dose-effect relationship of the increase in tumor cells in S-phase. In the second study, CPT-11 was given 24 h before GEM 1000 mg/m(2) per week for 2 weeks every 3 weeks. Doses of 20-80 mg/m(2) were explored to establish the MTD and DLT and to study tumor cell S-phase in selected patients. CPT-11 80 mg/m(2) produced a mean increase in S-phase by IHC for cyclin A of 137%. Lesser increases were seen with 40 and 60 mg/m(2). CPT-11 followed 24 h later by 5-FU 400 mg/m(2) per week for 4 weeks was well tolerated. In the study of CPT-11 followed by GEM 1000 mg/m(2), 60 mg/m(2) of CPT-11 was the MTD.

Effect of doxorubicin, daunorubicin, and idarubicin on the growth rate of human leukemia cells (K562) studied with image analysis.

The effects of doxorubicin, daunorubicin, and idarubicin on the growth of K562 cells were investigated by monitoring the formation of individual colonies in semi-solid culture employing an automated image analysis system. Drug effects were evaluated using short-term exposure (2 h) at clinically achievable drug plasma concentrations. Following drug exposure, heterogeneity in growth patterns was observed which was categorized into three distinct types; (1) continuous growth at a decreased growth rate; (2) limited growth; (3) no growth. In addition to a concentration-dependent decrease of the growth fraction, a reduction of the growth rate of the continuously growing colonies was observed. At equitoxic drug concentrations, comparable changes in growth rate were observed for each of the three anthracyclines studied. It is demonstrated that the fraction of cells which escape drug-induced growth arrest with therapeutically achievable drug concentrations display a significant decrease in growth rate.

Persistent induction of apoptosis and suppression of mitosis as the basis for curative therapy with S-1, an oral 5-fluorouracil prodrug in a colorectal tumor model.

In an effort to improve the therapeutic selectivity of 5-fluorouracil (FUra) against colorectal cancer, S-1, a combination agent including a prodrug of FUra with two modulators, was recently developed by Taiho Pharmaceuticals Co. S-1 is a combination of tegafur (FT), 5-chloro-2,4-hydroxypyridine, and potassium oxonate in the molar ratio of 1.0:0.4:1.0, with the latter two components as inhibitors of dihydropyrimidine dehydrogenase and phosphoribosylpyrophosphate transferase, respectively. In this study, the therapeutic selectivity and efficacy of S-1 (oral) was compared with FT (oral) and FUra (i.v. infusion) in rats bearing advanced colorectal cancer by using clinically relevant schedules. The maximum tolerated doses (MTDs) of S-1, FT, and FUra were 31.5, 200, and 25 mg/kg/d for 7 days and 22.5, 150, and 12.5 mg/kg/d for 28 days, respectively. The therapeutic index of S-1 was 4- to 5-fold higher than that of either FT or FUra. S-1 achieved 100% complete tumor regression (CR) at its MTD in both 7-day and 28-day schedules. Furthermore, the high incidences of stomatitis, alopecia, and diarrhea observed with FUra and FT, were not observed with S-1. In an attempt to understand the basis for the observed superior therapeutic selectivity with S-1, we studied pharmacokinetic analysis of FUra, drug-induced apoptosis, suppression of mitosis, and inhibition of thymidylate synthase (TS) after S-1, FUra, or FT administration. The peak plasma FUra concentrations derived from FUra or S-1 (FT) at comparable MTDs were similar, but the plasma level of FUra was higher with S-1 than with FUra. Induction of high and sustained apoptosis was achieved with S-1. Although the initial level of apoptosis induced by FUra was comparable to S-1, it was not sustained. The sustained level of apoptosis appears to correlate with tumor growth inhibition. Mitotic figures were more greatly suppressed with S-1 treatment than with FUra. Studies on TS inhibition indicated that, although both S-1 and FUra caused a 4- to 6-fold induction of total TS protein, single oral administration of S-1 was superior to 24-h infusion of FUra in suppressing free TS. The data are consistent with the observation that the therapeutic efficacy of S-1 (100% cure) over FUra is associated with high and sustained levels of drug-induced apoptosis, greater suppression of mitosis, and inhibition of free TS in tumor tissues.

Characterization of tumor cell heterogeneity of a murine leukemia cell line (L1210) in response to arabinosylcytosine: quantitation using a computerized image analysis system.

Cytosine arabinoside (Ara-C) is one of the most effective drugs in inducing remission in acute nonlymphocytic leukemia (ANLL) patients. However, the high recurrence rate indicates that a subpopulation of leukemic cells escapes drug effect. This cellular heterogeneity in drug response may play a major role in chemotherapeutic outcome. We have recently developed the individual colony-formation assay (ICFA) to study drug effects on the kinetics of proliferation of individual cells and their progeny. Thus parameters of proliferation are calculated for individual colonies. Three categories of drug responses were defined, including immediate growth cessation, delayed growth cessation (growth stops several days after drug exposure) and growth slowdown (logarithmic growth at a reduced rate compared to control). In the experiments included in this report, murine leukemia (L1210) cells were exposed to various concentrations of Ara-C for 1, 6 or 24 hours, and their responses quantified. Regardless of the Ara-C concentration or exposure time, subpopulations of cells were observed in each of the three response categories: immediate or delayed arrest or growth slowdown. As expected, the fraction of cells exhibiting immediate growth cessation generally increased with increasing drug dose and was markedly increased with longer exposure time. Delayed arrest was most prevalent at intermediate drug concentrations at all exposure times. If exposure was limited to 1 hour, at least 30% of cells continued to grow, although at a reduced rate (71% control rate after exposure to 1 mM Ara-C). This limited effect was paralleled by saturation of Ara-C triphosphate (Ara-CTP) formation. Six-hour exposure left at least 6.4% of cells growing, with an average rate of 45% of control. Under these conditions, no saturation in Ara-CTP formation was observed. Even 24-hour exposure to 5 microM Ara-C left 4.8% of colonies growing, at 42% of control rate. Thus a subpopulation of cells continued to grow even after 24-hour exposure to a relatively high concentration of Ara-C. Surviving, but slowly growing, cells may represent a previously unrecognized population that may contribute to therapeutic failure.

Growth slow-down and growth arrest of human colon carcinoma cells HCT-8 in vitro after exposure to 5-fluoro-2'-deoxyuridine.

Cellular heterogeneity in drug response denotes a mixed response among individual cells in a drug treated population. Individual cell responses may be more complex than 'cell kill' and 'no response'. In this study we employed a colony formation assay and high-resolution image analysis to detect the various responses such as immediate and delayed cessation of growth, growth delay and growth slow-down, at the level of the individual colony. The evaluation was carried out using a human ileocaecal adenocarcinoma cell line (HCT-8) and the anti-tumour agent 5-fluoro-2'-deoxyuridine (FdUrd). In the presence of a drug concentration which, in standard monolayer assays, inhibits the growth to about 50% (IC50) only about 20% of the colonies ceased to grow and the remaining colonies grew at a growth rate of about 70% of control. At an FdUrd concentration which, in standard monolayer assays, reduced growth by > 90% (> IC90), about 50% of the cells grew, with growth rates of about 30% of control. The slowing of growth, most prominent at lower drug concentrations, should be considered in determining mechanisms of drug action at the individual cell level. In clinical situations in which high drug doses are precluded by toxicity to normal tissues, growth slow-down may play a significant role in tumour response.

Mechanisms of acquired resistance to modulation of 5-fluorouracil by leucovorin in HCT-8 human ileocecal carcinoma cells.

Repeated (10x) exposure of HCT-8 human ileocecal carcinoma cells to 5-fluorouracil (5-FU) for 2 or 72 hr, both incubations in the continuous presence of 20 microM leucovorin (LV), yielded two stable modulation-resistant sublines, FL2h and FL72h. Although LV potentiated growth inhibition by 5-FU 2-fold in parental HCT-8 cells, it did not potentiate the effect of 5-FU in the FL2h or FL72h sublines. LV modulation of 5-fluorodeoxyuridine (5-FdUrd) was also reduced (FL72h) or eliminated (FL2h). In the FL2h and FL72h sublines, the level of thymidylate synthase (TS) protein and TS activity in cell extracts, TS activity in situ, the rate of cellular uptake and metabolism of LV, and the level of 5-FU incorporation into total cellular RNA were similar to those in parental HCT-8 cells. However, LV significantly (P < 0.01) potentiated the inhibition of TS activity in situ in HCT-8 cells at 24 hr after a 2-hr treatment with either 5-FU or 5-FdUrd, but had no such activity in the FL2h and FL72h sublines (P > 0.1). Resistance to modulation of 5-FU by LV was associated with the inability of LV to increase the formation of intracellular TS-FdUMP-methylenetetrahydrofolate ternary complexes, and these complexes dissociated more rapidly (T1/2 > 1.5- to 3-fold faster) in the presence of different concentrations of 5,10-methylenetetrahydropteroylpentaglutamate. Thus, decreased stability of ternary complexes appears to be the mechanism of acquired resistance to the LV modulation of fluoropyrimidine cytotoxicity, possibly due to mutation(s) of TS in these two modulation-resistant HCT-8 sublines.

Cellular heterogeneity in DNA damage and growth inhibition induced by ICI D1694, thymidylate synthase inhibitor, using single cell assays.

Heterogeneity in the response of the HCT-8 (human ileocecal adenocarcinoma) tumor cell line to a new thymidylate synthase inhibitor, ICI D1694, was investigated in terms of induction of DNA single-strand breaks and cytotoxicity, applying the single cell alkaline gel (SCG) electrophoresis assay and the individual colony formation assay (iCFA), respectively. ICI D1694 induced maximal total DNA single-strand breaks 24 hr after a 2-hr drug exposure with incomplete repair by 72 hr. The level of DNA damage was concentration dependent and paralleled cellular growth inhibition in vitro. The proportion of cells with DNA damage and the extent of DNA single-strand breaks increased with drug concentration. At 1 microM ICI D1694 (IC95), a significant level of DNA damage was detected in 58% of the cells; however, 25% of the cells had little or no damage. Using the iCFA system, it was observed that with 1 microM ICI D1694, only 2.6% of the seeded cells maintained a colony growth rate similar to that of the control colonies, and 22% of the cells were growing significantly more slowly. In conclusion, the SCG assay and the iCFA identified subpopulations of cells that were unaffected by ICI D1694. Although these cells represented only a small proportion of the total cell population, this phenomenon of heterogeneity in response to ICI D1694 might limit its therapeutic efficacy.

Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine.

A 5-fluoro-2'-deoxyuridine (FdUrd)-resistant subclone (Fd9XR) of HCT-8 (human ileocecal carcinoma) cells was established by two schedules of drug exposure. Initially, cells were exposed to short-term (3 hr) 100 nM FdUrd repeatedly (9 cycles over 8 months), and cells were then exposed to 10 nM FdUrd continuously. During this latter stage, a colony (Fd9XR) with fast growth rate was isolated, expanded, and characterized with respect to mechanisms of resistance to FdUrd and cross-resistance to other chemotherapeutic agents. Fd9XR cells were 1000-fold resistant to FdURD, but 3-fold more sensitive to 5-fluorouracil (FUra) than HCT-8 cells. After a 3-hr treatment with FdUrd, Fd9XR cells accumulated 6630-, 69-, and 3.7-fold less fluorodeoxyuridylate (FdUMP), fluorouridine triphosphate (FUTP) and acid-insoluble materials, respectively, than HCT-8 cells. However, when FUra was substituted for FdUrd, Fd9XR cells accumulated 9.2-, 3.1-, and 2.3-fold more FdUMP, FUTP and acid-insoluble materials, respectively, than HCT-8 cells. Fd9XR and HCT-8 were similar in their growth rates, combined pools of 5,10-methylenetetrahydrofolates (5,10-CH2H4PteGlun) and tetrahydrofolates (H4PTeGlun), thymidine phosphorylase (TP) activity, and level and activity of thymidylate synthase (TS). In contrast, thymidine kinase (TK) activity of Fd9XR was 0.23 and 0.35% of that of HCT-8, for thymidine (dThd) and FdUrd as substrates, respectively. Furthermore, Fd9XR cells exhibited greater sensitivity to the antifolate TS inhibitor ICI D1694 and to methotrexate (MTX) than HCT-8 cells. In addition, dThd alone and in combination with hypoxanthine did not offer any protection against the cytotoxic effect of ICI D1694 in Fd9XR cells. These results indicate that in Fd9XR cells (1) TK deficiency is the primary mechanism of resistance to FdUrd; (2) the greater sensitivity to FUra was associated with higher pools of FdUMP and FUTP with a subsequently higher level of incorporation into cellular RNA; and (3) antifolate compounds, e.g. ICI D1694 and MTX, could be useful agents in the treatment of FdUrd-resistant tumors associated with decreased TK activity and decreased capacity of utilizing dThd.

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin -6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034): comparison across sensitive and resistant human tumor cell lines.

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

Correlation of tumor-cell growth in four semisolid systems.

The correlation of the colony growth of cells disaggregated from human melanoma, sarcoma, lung, and ovarian carcinomas were studied in four different semisolid tissue culture assays: (a) the soft agar assay of Pluznik and Sachs; (b) the soft agar assay of Hamburger and Salmon; (c) the soft agar-methyl cellulose assay of Buick et al.; and (d) the methyl cellulose assay of Ogawa et al. There was no colony growth of tumor cells achieved in 15 of 15 cases assayed in Ogawa's methyl cellulose assay. The plating efficiency of the above mentioned tumors was similar in the assays of Pluznik and Sachs, Hamburger and Salmon, and Buick et al. However, the tumor take rate differed among these three systems. The assay of Buick et al. appears potentially useful for analysis of the biology of human tumors.

Limitations of drug sensitivity testing in soft agar for clinical management of patients with ovarian carcinoma.

Thirty specimens from 28 patients (15 previously treated, 13 previously untreated) with ovarian carcinoma were studied in a soft agar colony-forming assay to determine whether or not the assay could be useful for treatment planning. There were sufficient colonies for drug testing in 23 cases. An in vitro drug response was found in 12 of 106 drug tests. In vivo-in vitro correlations could be made for 28 drug trials in 16 patients. Eight patients were not evaluated for response because they were clinically disease free after debulking surgery. Single agents were evaluated in vitro, although most patients received combination chemotherapy. In 23 cases the tumor was resistant in vitro and in vivo. There were two false-negative and three false-positive results. Cell aggregates that may artificially increase growth rates and apparent in vitro drug resistance were a major problem technically. In view of the problems identified, the assay in its current form should not be used routinely to direct therapy.

Chromosomes and causation of human cancer and leukemia. XLIV. A method for chromosome analysis of solid tumors.

Mechanical and enzymatic disaggregation procedures have been utilized for the preparation of materials suitable for chromosome examination of tissues from various kinds of solid tumors. A great number of the cells disaggregated with the latter procedure were observed to attach to the bottom of culture flasks after 2 days in vitro; cells obtained with the former method were not attached. In general, the enzymatic procedure yielded a significantly larger number of viable cells that would undergo mitosis after short term culture and considerably improved the quality of banding as compared to the mechanical method. However, there were no significant differences among the basic karyotypes observed in the cells obtained by the mechanical approach versus those seen following the enzymatic method. This evidence, together with the high success rate of karyotyping in the enzyme-treated preparations, suggest that this should be the procedure of preference for cytogenetic examination of various tumors, particularly where in the past karyotypic examination has been very difficult, e.g., the early stages of tumor development.