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To clarify the inhibitory effect of telomere-mimic oligonucleotides on human cancer cell lines, we synthesized 18-mers (18T; n = 3), 24-mers (24T; n = 4) and 30-mers (30T; n = 5) of telomere-mimic phosphorothioate oligodeoxy nucleotides [5'-d(TTA GGG)n-3'] and examined their effects on the proliferation of human tumor cells by XTT assay. After 7 days of continuous exposure to 24T and 30T at concentrations ranging from 0.1 to 10 microM, concentration-dependent cell growth inhibition was observed in MCF-7 clone E3, ZR-75-1, MDA-MB 231, Colo 201 and WiDr. All of these cell lines highly expressed telomerase using the telomeric repeat amplification protocol. None of these tumor cell lines were affected by 18T. In MCF-7, ZR-75-1 and Colo 201 cell lines, a more than 50% growth inhibition was obtained by 3 microM of 24T and 30T whereas, in MDA-MB 231 and WiDr cell lines, cell growth inhibition was less than 50%. 30T was more effective than 24T. Estrogen-dependent growth of both MCF-7 and ZR-75-1 was inhibited by 3 microM of 24T and 30T, however, in the absence of estrogen, no growth inhibition was seen. The MCF-10A cell line, which was developed from normal human breast tissue and expressed telomerase only weakly, was inhibited by 10 microM of 18T. In conclusion, these observations indicate that S-ODNs inhibit tumor growth in cell lines expressing telomerase in a concentration-dependent manner and that cell growth inhibition is dependent on the length of S-ODNs. In addition, the short-length S-ODNs may inhibit growth of cells weakly expressing telomerase, but not of cells with high telomerase expression.

The combination of pyrazofurin (PF) and 5-azacytidine (5-aza-CR) was evaluated in 12 patients with neoplastic diseases who had failed conventional therapies. They included 6 patients with acute myelocytic leukemia (AML) and 6 with various carcinomas. PF was given by i.v. bolus at doses ranging from 25 to 200 mg/m2 followed by 5-aza-CR given by continuous 24-hour infusion at doses ranging from 30 to 120 mg/m2/24 h during 40-ic diseases who had failed conventional therapies. They included 6 patients with acute myelocytic leukemia (AML) and 6 with various carcinomas. PF was given by i.v. bolus at doses ranging from 25 to 200 mg/m2 followed by 5-aza-CR given by continuous 24-hour infusion at doses ranging from 30 to 120 mg/m2/24 h during 40-ic diseases who had failed conventional therapies. They included 6 patients with acute myelocytic leukemia (AML) and 6 with various carcinomas. PF was given by i.v. bolus at doses ranging from 25 to 200 mg/m2 followed by 5-aza-CR given by continuous 24-hour infusion at doses ranging from 30 to 120 mg/m2/24 h during 40--120 h. Dose-limiting toxicity was mucocutaneous, which appeared in 11/17 courses. Skin rash was similar to that produced by PF alone, but was present after much lower doses of PF when followed by 5-aza-CR. Moderate and reversible thrombocytopenia and leukopenia occurred in 3/6 patients with carcinoma. No objective response was seen in patients with carcinoma, and no marrow remission occurred in patients with AML. 24-Hour urinary excretion of orotidine and oroticacid were measured during 11 courses in 8 patients, and were not much different than those obtained previously with PF alone. The occurrence of mucocutaneous toxicity precluded the use of higher doses of PF and 5-aza-CR, which might have been therapeutically more active.

The new synthetic tripeptide, p-fluoro-L-phenylalanyl-m-bis-(2-chloroethyl)-amino-L-phenylalanyl-methi onine ethylester hydrochloride, PTT.119, an alkylating agent, is currently undergoing preclinical trials as an antineoplastic agent. The molecular composition, C29H39N4O4SCl2F, was confirmed by desorption chemical ionization mass spectrometry with accurate mass measurement. A high-performance liquid chromatographic technique was developed for the quantification of PTT.119 in cell culture medium and serum. Incubation of 5 X 10(5) mammary tumor cells (MJY-alpha)/ml tissue culture medium with 25 micrograms PTT.119/ml for 60 min (37 degrees C) removed 68% of the tripeptide from the medium. This corresponds to an uptake of 51 fmol PTT.119/tumor cell. Cell death, assessed 5 days after treatment, was directly proportional to the time-dependent removal of PTT.119 from the cell culture medium.

Inhibitory effects of two human interferon preparations, leukocyte interferon (Le-IF) and fibroblast interferon (F-IF), on granulocytic progenitor cells (CFU-C) from hematologically normal cancer patients and from patients with chronic myelogenous leukemia were evaluated. There was a wide variation in sensitivity of CFU-C to both Le-IF and F-IF. F-IF was mor inhibitory against CFU-C than le-If. Normal CFU-C and chronic myelogenous leukemia CFU-C were equally inhibited by both interferons. Effects of both interferons were neutralized by corresponding specific antisera but not by the other antisera. These observations confirmed that differences in immunogenicity of the interferons may attend their different origins.

Delivery of the bifunctional alkylating agent, PTT.119 [p-F-L-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met-ethoxy-HCl], into tumor cells is significantly greater compared to L-phenylalanine mustard (L-PAM) as demonstrated by the 2-fold reduction in PTT.119 dosage required to reduce the viable L1210 cell fraction by 50% (TCD50). This increased uptake and consequent cytolytic efficacy observed in Dulbecco's phosphate buffer was more apparent in culture medium; under this physiologic condition the TCD50 concentration of PTT.119 was 5 times lower than L-PAM. PTT.119 entry into leukemia cells was examined using competition transport assays assessing the ability of the tripeptide to compete with various amino acids and nonmetabolizable substrates for carrier receptors of the L, A and ASC transport systems. A 1-min exposure to a 1- to 50-fold excess of PTT.119 prior to addition of radiolabeled substrates significantly reduced within 60 s both sodium-dependent and sodium-independent uptake of leucine, methionine, threonine and alpha-[1-14C]-aminoisobutyric acid (AIB), but not MeAIB. In complimentary studies, L1210 cells were protected from PTT.119 cytolysis by an 8,000-fold excess of AIB, whereas beta-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) only abrogated tripeptide cytotoxicity by 95-97% even at BCH:PTT.119 ratios of 200,000. Leucine and methionine protection were significantly less effective; the TCD50 of leucine and methionine were 3.23 and 2.4 microM, respectively, compared to 11.41 microM for AIB and 7.96 microM for BCH. In addition, MeAIB and phenylalanine were totally unable to protect L1210 cells from PTT.119-induced cytolysis. The data indicate that L121 cells actively transport PTT.119 primarily by the BCH-sensitive, AIB-sensitive, MeAIB-insensitive L carrier system. A second, BCH-insensitive, AIB-sensitive and MeAIB-insensitive carrier which is also involved in tripeptide uptake is probably the ASC system.

We studied the effects of anticancer agents on the inhibition of cell shedding from the surface of multicellular tumor spheroids (MTS). MTS were produced from 2 human tumor cell lines; one melanoma and the other squamous cell lung cancer, by using liquid overlay culture technique. The cell shedding from the melanoma MTS was approximately 10-fold higher than the squamous cell carcinoma MTS. In the melanoma MTS, all 3 drugs studied - vincristine (VCR), doxorubicin (ADR) and cisplatin (DDP)-inhibited cell shedding and the degree of inhibition of cell shedding was drug concentration related. In the squamous cell carcinoma MTS, VCR was as active in inhibiting cell shedding as in the melanoma MTS, but ADR and DDP were less efficacious. When effects on cell shedding were compared with those on cell lethality, VCR produced inhibition of cell shedding at much lower concentrations than those producing cell kill effects. ADR and DDP produced cell lethality as effective as, or more effective than, inhibition of cell shedding. These data seem to parallel known effects of these agents on cell kill and inhibition of metastases. MTS may serve as an in vitro model for the study of cell shedding and metastasis.