Sensitivity of proliferating cultured murine pancreatic tumor cells to selected antitumor agents.

Cultured cell populations derived from the refractory murine pancreatic ductal adenocarcinoma (Panc 02) were propagated in modified Eagle's minimal essential medium supplemented with 20% fetal bovine serum and had a doubling time of 19.1 +/- 4.7 hours (mean +/- SD). The Panc 02 cell populations were tested against 8 antitumor agents and exhibited different sensitivities to the agents in a 24-hour growth-inhibition assay. The concentration that inhibits the growth of the test culture by 50% relative to the growth of the control culture (IC50) in micromolars was determined for each agent. The IC50 values were: doxorubicin (ADR), 0.055; vincristine (VCR), 0.042; 5-fluorouracil, 1.92; cytarabine, 5.35; melphalan, 10.5; cisplatin, 17.0; carmustine (BCNU), 46.2; and lomustine (CCNU), 52.6. These IC50's were estimated to be pharmacologically attainable concentrations in mice. On a micromolar basis, the Panc 02 cells were the most sensitive to VCR and ADR and the least sensitive to BCNU and CCNU. By the use of a colony-forming assay and a 24-hour exposure period and the evaluation of each agent at 1/3 X IC50, 1 X IC50, and 3 X IC50, the degree of cell killing was greater than predicted on the basis of the IC50's determined in the growth-inhibition assay. The use of a 1-hour exposure period resulted in a very minimal reduction in viability of the cell populations except for BCNU and CCNU. It was concluded that the degree of cell killing was a function of drug concentration and time of exposure and that the pancreatic tumor in vivo should be sensitive to these agents, provided effective concentrations and exposure periods can be achieved at the tumor target sites.

Inhibition and reversal by beta-retinoic acid of hyperplasia induced in cultured mouse prostate tissue by 3-methylcholanthrene or N-methyl-N'-nitro-N-nitrosoguanidine.

The effect of beta-retinoic acid (RA) on carcinogen-induced hyperplasia was studied in organ cultures of mouse prostate gland. 3-Methylcholanthrene (MCA), requiring metabolic activation, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), not requiring activation, were used to induce hyperplastic changes. Treatment of cultures with MCA or MNNG stimulated cell proliferation and caused the alveolar epithelium to become hyperplastic. The development of this hyperplasia was inhibited when RA was added simultaneously with MCA or MNNG. However, RA had no significant effect on cell proliferation in untreated control cultures. Elimination of carcinogen from the hyperplastic cultures after 8 days of treatment did not reverse hyperplasia of the alveolar epithelium. When the withdrawal of MCA or MNNG was followed by treatment of the cultures with RA, hyperplasia was markedly reversed within 96 hours. Thus RA actively inhibited and reversed the effect of MCA and MNNG, two carcinogens that may have different mechanisms of action.

Reversal by vitamin A analogues (retinoids) of hyperplasia induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse prostate organ cultures.

The antihyperplastic activity of beta-retinoic acid (RA) and nine synthetic analogues (retinoids) was examined in organ cultures of mouse prostate made hyperplastic by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After 8 or 10 days, when most explants developed hyperplasia, the carcinogen was withdrawn and explants were incubated in control medium and medium containing different concentrations of a retinoid. The antimitotic activity of retinoids was compared with that of RA. Different retinoids produced variable degrees of mitotic inhibition in the hyperplastic prostate epithelium. The methylketo cyclopentenyl and 1-methoxyethyl cyclopentenyl analogues of RA were at least 50-fold more active than RA in reversing MNNG-induced hyperplasia. The trimethylmethoxyphenyl analogue of RA and retinyl methyl ether were significantly more active than RA. Three analogues, N-acetyiretinylamine, retinal acetyl hydrazone, and retinal oxime, were as active as RA. The chlorotrimethylphenyl analogue showed less activity than RA, and alpha-retinyl acetate was completely devoid of mitotic inhibitory activity.

Resistance and cross-resistance of cultured leukemia P388 cells to vincristine, adriamycin, adriamycin analogs, and actinomycin D.

Cultured P388 cells derived from leukemia P388 passaged in vivo in (C57BL X DBA/2)F1 mice were approximately one-tenth less sensitive to N-trifluoroacetyladriamycin-14-valerate (AD32) and cinerubin A, inasmuch as the concentrations of these agents had to be increased tenfold to produce a reduction in cell viability of the same order of magnitude as that produced by adriamycin, daunomycin, or actinomycin D. Cultured P388 cells derived from resistant cell populations developed in vivo retained their resistance to vincristine. These resistant cells exhibited cross-resistance to actinomycin D, adriamycin, daunomycin, and AD32, but cross-resistance to cinerubin A could not be detected. Data also showed that the vincristine-resistant cells were more sensitive to AD32 (2.8 x 10(-6) M) than to adriamycin (3.4 x 10(-6) M) during a 6-hour exposure of cells to agents.

Cross-resistance of cultured murine leukemia vincristine-resistant P388 cells to vinblastine, vindesine, and bis (N-ethylidene vindesine) disulfide, disulfate.

Cultured P388 leukemia cells derived from vincristine (VCR)-resistant cell populations developed in vivo in (C57BL X DBA/2)F1 mice (P388/VCR) were cross-resistant to vinblastine (VLB), vindesine (VDS), and bis(N-ethylidene vindesine)disulfide, disulfate (bis-VDS). Cross-resistance was a function of concentration and time of exposure. Cultured P388/VCR cells were more sensitive to 6.1x10-5Mbis- VDS than to 6.1x10-5MVDS or to 6.1x10-5MVLB. Cultured P388 cells derived from VCR-sensitive P388 leukemia passaged in vivo (P388/0)were significantly more sensitive to VDS and bis-VDS than to VLB. This degree of difference in sensitivity was also a function of concentration and time of exposure.

Evaluation of vitamin A analogs in modulating epithelial differentiation of 13-day chick embryo metatarsal skin explants.

Seventeen vitamin A compounds were evaluated in organ culture for activity in altering epithelial differentiation of metatarsal skin explants from 13-day-old chick embryos. The explants keratinized in 6 to 8 days and, when cultured in the presence of beta-retinoic acid (RA), inhibition of keratinization occurred and a mucous metaplasia developed. A cyclopentenyl analog of retinoic acid was approximately 10-fold more effective than RA in producing mucous metaplasia. Six other analogs exhibited about the same activity as RA: trimethylmethoxyphenyl analog of retinoic acids, alpha-retinoic acid, 13-cis-retinoic acid, methyl retinoate, ethyl retinoate, and N-ethylretinamide. The following 5 vitamin A compounds were about one-fourth as effective as RA: the trimethylmethoxyphenyl analog of ethylretinamide, the phenyl analog of retinoic acid, the trimethylmethoxyphenyl analog of ethyl retinoate, beta-retinyl acetate, and retinol. The furyl analog of retinoic acid and N,N-diethylretinamide were approximately one-tenth and one-fifteenth less effective than RA in inhibiting keratinization. The analog, alpha-retinyl acetate, was about one-hundredth as effective as RA and the pyridyl analog of retinoic acid (2.5 X 10(-5) M) did not inhibit keratinization. Since the property of altering epithelial differentiation may be a fundamental requirement for the prophylaxis and/or treatment of malignant epithelial lesions, this system can be used to determine whether the new synthetic analogs of vitamin A are active in modulating epithelial differentiation.

Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice.

Following implant of cotton thread-carrying 3-methyl-cholanthrene into the pancreas tissue of 90 C57BL/6 and 60 BALB/c mice, 13 developed ductal adenocarcinomas. Two of these tumors, both of C57BL/6 origin (Panc 02 and 03), were established in serial s.c. transplant. Panc 02 was treated with 37 different anticancer drugs representing all of the chemical and functional classes of clinically useful anticancer agents including alkylating agents, antimetabolites, agents that bind to or cause scission of DNA, and others that inhibit mitosis or inhibit protein synthesis. When drug treatment was started within 3 to 4 days after tumor implant, Panc 02 showed only limited response to treatment with two nitrosoureas, [N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N- nitrosourea, monohydrochloride and N-(2-chloroethyl)-N'-(2,6-dioxo-3-piperdinyl)-N-nitrosourea)], and N-phosphonacetyl-L-aspartate. Drug response of Panc 03 was determined only with Adriamycin, 5-fluorouracil, cyclophosphamide, cis-(SP-4-2)-diamminedichloroplatinum, or N,N'-bis(2-chloroethyl)-N-nitrosourea. When drug treatment was started 3 days after tumor implant, high cure rates were obtained with Adriamycin treatment, and limited therapeutic responses were seen to treatment with cis-diamminedichloroplatinum or cyclophosphamide. A comparison of the biological characteristics and drug responsiveness of Panc 02 and Panc 03 with those of a number of other transplantable tumors of mice is reported.

Effect of retinoids on the differentiation of chick embryo metatarsal skin explants.

Twelve retinoids were evaluated in organ culture for activity in modulating epithelial differentiation of metatarsal skin explants from 13-day chick embryos. The epithelium differentiated into a squamous, keratinizing epidermis; but, in the presence of active retinoids, keratinization was inhibited, and a mucous metaplasia developed. The methyl-keto and 1-methoxyethyl cyclopentenyl analogs of retinoic acid were about tenfold more effective than retinoic acid in altering epithelial differentiation. The dichlorophenyl analog exhibited about the same activity as retinoic acid. The following analogs were one-half to one-third as effective as retinoic acid in inhibiting keratinization: the chlorotrimethylphenyl analog of retinoic acid and the 13-cis, 10-fluoro analog of trimethylmethoxyphenyl methyl retinoate. The other 7 retinoids were essentially not active at the concentration tested (1.4--2.0 x 10(-5) M). The activity of synthetic retinoids in altering epithelial differentiation may be related to their ability to affect or treat epithelial lesions provided that modification of the retinoid molecule can enhance its activity and decrease toxicity.

Etoposide-resistant human colon and lung adenocarcinoma cell lines exhibit sensitivity to homoharringtonine.

Human colon (HCT116/VP48) and lung (A549B/VP29) adenocarcinoma cell lines selected for resistance to etoposide exhibited modified patterns of multi-drug resistance (MDR) that included a differential sensitivity to other DNA topoisomerase II inhibitors and to the plant alkaloids homoharringtonine, vinblastine, and vincristine. The resistance and cross-resistance drug phenotype of the A549B/VP29 cell line was different from that of the HCT116/VP48 cell line. The HCT116/VP48 cell line was 50-fold resistant to etoposide and 30-fold resistant to teniposide. The degree of resistance to other DNA topoisomerase II inhibitors was of a lower magnitude: Adriamycin, 9-fold; daunomycin, 3-fold; 4'-[(9-acridinyl)-amino]-methanesulfone-m-anisidide (m-AMSA), 3-fold; and actinomycin D, 6-fold. The HCT 116/VP48 cell line exhibited a 7-fold resistance to vincristine and a 2-fold resistance to vinblastine but was sensitive to homo-harringtonine. The A549B/VP29 cell line was 5-fold resistant to etoposide and 2-fold resistant to teniposide. The A549B/VP29 cell line exhibited a 2-fold resistance to Adriamycin but was sensitive to daunomycin and showed a 3-fold resistance to m-AMSA. This cell line was sensitive to actinomycin D. The A549B/VP29 cell line was 2-fold resistant to vinblastine and sensitive to homoharringtonine. Both cell lines (HCT116/VP48 and A549/VP29) exhibited no amplification of the human mdr1 DNA sequence, the 4.3-kb P-glycoprotein transcript, or the membrane P-glycoprotein. The sensitivity of cells exhibiting an MDR phenotype not mediated by P-glycoprotein suggests a potential use for homoharringtonine in treating tumors with this type of drug resistance.

Effect of homoharringtonine on the viability of murine leukemia P388 cells resistant to either adriamycin, vincristine, or 1-beta-D-arabinofuranosylcytosine.

Cultured murine leukemia P388 cell populations were derived from P388 cells resistant to vincristine (P388/VCR), adriamycin (P388/ADR), and 1-beta-D-arabinofuranosylcytosine (P388/ARA-C) that were developed in vivo and to the parental drug-sensitive cells (P388/O) that were passaged in vivo. The doubling times of the cultured cell populations (mean +/- SD) between cell densities of 5 x 10(4) and 1 x 10(6) cells/ml were 14.2 +/- 2 h (P388/O), 16.5 +/- 1.9 h (P388/VCR), 16.9 +/- 1.2 h (P388/ADR), and 15.0 +/- 1.4 h (P388/ARA-C). Exponentially proliferating cultured cell populations were exposed to selected homoharringtonine (HHT) concentrations for 24 h and the surviving cell fractions were determined by colony formation in semisolid medium. The results, based on differential sensitivity of the cell populations to HHT, indicated that cultured P388/VCR cells were cross-resistant to 0.018-1.8 micrograms/ml HHT, P388/ADR cells were cross-resistant to 0.058-1.8 micrograms/ml HHT, and P388/ARA-C cells were collaterally sensitive to 0.09-0.36 micrograms/ml HHT. The results with the cultured P388/VCR, P388/ADR, P388/ARA-C, and P388/O cell populations were confirmed in animal experiments. CD2F1 mice bearing intraperitoneal (i.p.) implants of 1 x 10(6) P388/VCR, P388/ADR, P388/ARA-C, or P388/O leukemia cells were given HHT i.p. qd on days 1-9 postimplantation. Optimal treatment (less than or equal to LD10) produced in vivo cell kills of 2 to 3 log10 units in P388/O and about 7 log10 units in P388/ARA-C, whereas P388/VCR and P388/ADR cells actually increased by 1-2 log10 units during treatment. The results of this study indicate that cross-resistance (P388/VCR and P388/ADR) or collateral sensitivity to HHT (P388/ARA-C) is a function of the cellular properties of the target tumor cell populations that is independent of host factors.

Evaluation of trans-tetrachloro-1,2-diaminocyclohexane platinum (IV) in murine leukemia L1210 resistant and sensitive to cis-diamminedichloroplatinum (II).

trans-Tetrachloro-1,2-diaminocyclohexane platinum (IV) (tetraplatin) was therapeutically effective in mice bearing leukemia L1210 resistant (L1210/DDPt) or sensitive (L1210/0) to cis-diamminedichloroplatinum (II) (cisplatin). Furthermore, the sensitivity of cultured L1210/DDPt and L1210/0 cell populations to tetraplatin, cisplatin, and dichloro-trans-dihydroxyisopropylamine platinum (IV) (CHIP) was a function of the concentrations used for each compound. The relative degree of sensitivity between cultured L1210/DDPt and L1210/0 cells for each compound on the basis of the LC99 (the concentration of each compound required to reduce the number of viable cells by 99% in each cell line) was 3-fold for cisplatin, 2-fold for tetraplatin, and 3-fold for CHIP; thus the cultured L1210/0 cells exhibited a greater degree of sensitivity than the L1210/DDPt cells to the platinum compounds. The data indicate that if reduction of platinum IV compounds to platinum II compounds or metabolites is required for antitumor activity, then the cultured L1210 cells are capable of this bioreduction independently of any host factors.

Induction of hyperplasia and anaplasia by carcinogens in organ cultures of mouse prostate.

In an effort to establish a test system to examine the carcinogenic potential of chemicals, mouse prostate explants were maintained as organ cultures and the effects of carcinogenic and noncarcinogenic compounds were examined at various intervals after treatment. The degree of hyperplasia produced by a compound was determined by the colcemid metaphase arrest technique. Extensive hyperplasia of the prostatic epithelium occurred at 8 days after treatment with 3-methylcholanthrene, the 11-12 epoxide of methylcholanthrene, benzo(a)pyrene and N-methyl-N-nitro-N-nitrosoguanidine. At 12 days most carcinogen-treated explants were anaplastic. The noncarcinogenic compounds, pyrene and phenanthrene, did not produce a mitotic stimulatory effect on the epithelium of the explants. The data suggest that the organ culture system of mouse prostate may be employed as a test system to obtain preliminary information regarding the cardinogenicity of a compound.

Kinetics of reduction in viability of cultured leukemia L1210 cells exposed to purine analogs.

Rates of reduction in viability and degree of cell killing were relatively independent of concentrations when replicating cultured L1210 cells were exposed to increasing concentrations of 6-thioguanine, 6-methylthiopurine ribonucleoside, 9-beta-D-arabinofuranosyl-9H-purine-6-thiol, or 9-ethyl-6-thiopurine. The relative lack of dependence of cell killing rate on cytotoxic concentrations suggest (a) that these agents may be only effective aganinst proliferating cells, and (b) that only limited therapeutic advantage can be gainged by increasing their concentration beyond a minimum effective level. In contrast, the rate and degree of cell killing were dependent upon concentrations when cells were exposed to increasing amounts of 4-aminopyrrolo(2,3-d)pyrimidine-beta-D-ribofuranoside or to 2-fluoroadenosine, indicating that these analogs are not cell-cycle-stage-specific and that nonreplicating cell populations may be sensitive to them.

Factors affecting the persistence of Staphylococcus aureus on fabrics.

The persistence of Staphylococcus aureus (Smith) on wool blanket, wool gabardine, cotton sheeting, cotton knit jersey, cotton terry cloth, and cotton wash-and-wear fabrics was studied. The fabrics were exposed to bacterial populations by three methods: direct contact, aerosol, and a lyophilized mixture of bacteria and dust having a high content of textile fibers. The contaminated fabrics were held in 35 or 78% relative humidities at 25 C. In general, the persistence time of S. aureus populations on fabrics held in 35% relative humidity was substantially longer when the fabrics were contaminated by exposure to aerosolized cultures or to dust containing bacteria than when contaminated by direct contact. In a 78% relative humidity, bacterial populations on the fabrics persisted for substantially shorter periods of time regardless of the mode of contamination or fabric type. Cotton wash-and-wear fabric (treated with a modified triazone resin) was the material on which populations of S. aureus persisted for the shortest time. This organism retained its virulence for Swiss mice after being recovered from wool gabardine swatches held 4 weeks in 35% relative humidity and 6 weeks in 78% relative humidity.

Persistence of Salmonella typhimurium on fabrics.

The persistence of Salmonella typhimurium (V-31) on wool blanket, wool gabardine, cotton sheeting, cotton knit jersey, cotton terry cloth, and cotton wash-and-wear fabrics was studied. Three methods of exposure were employed to contaminate the fabrics: direct contact, aerosol, and a lyophilized mixture of bacteria and dust having a high content of textile fibers. After contamination, the fabrics were held in 35 or 78% relative humidity at 25 C. The persistence time of S. typhimurium on fabrics held in 35% relative humidity was substantially longer when the fabrics were contaminated by direct contact or by exposure to dust containing bacteria than when contaminated by exposure to aerosolized cultures. Viable bacterial populations persisted for 24 weeks at relatively high population densities on swatches of wool gabardine, cotton sheeting, cotton knit jersey, and cotton terry cloth exposed by direct contact and held in a humidity of 35%. In 78% humidity, bacterial populations persisted on the fabrics for relatively shorter periods of time regardless of the mode of contamination or fabric type. This organism retained its virulence for Swiss mice after being recovered from wool gabardine swatches held 8 weeks in humidities of 35 or 78% and from cotton terry cloth swatches held 6 weeks in the same humidities.

Potentially infectious agents associated with shearling bedpads: effect of laundering with detergent-disinfectant combinations on Staphylococcus aureus and Pseudomonas aeruginosa.

Glutaraldehyde-tanned woolskins which are used as bedpads to prevent decubitus ulcers were contaminated with Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442). Two methods of exposure, direct contact and aerosol, were used in separate experiments. Attempts were made to decrease the bacterial population placed on the woolskins by laundering them in a quaternary ammonium disinfectant, a phenolic disinfectant, or alkalinized glutaraldehyde, in combination with an anionic or nonionic detergent. The effect of a commercial detergent-sanitizer was also studied. Bacterial populations were significantly reduced in all experiments, but only laundering in glutaraldehyde in combination with either detergent resulted in maximum removal of bacteria. Viable bacteria were usually not detected in the rinse water (<1 viable organism/5 ml of rinse water).