Collateral methotrexate resistance in cultured human head and neck carcinoma cells selected for resistance to cis-diamminedichloroplatinum(II).

A human head and neck squamous cell carcinoma line (SCC25) derived from a patient with no prior history of radiotherapy or chemotherapy was made resistant to cis-diamminedichloroplatinum(II) (CDDP) by continuous escalation of weekly 30-min pulses of the CDDP from 0 to 0.2 mM over 20 months and then cloned and pulsed weekly with 0.2 mM CDDP for another 20 months. This afforded a resistant subline, SCC25/CP[1], with an IC50 for CDDP 12-fold higher than that of the parental cells. The SCC25/CP[1] cells unexpectedly proved to be cross-resistant to methotrexate (MTX) (24-fold for 30-min treatment and 8-fold for continuous treatment). Resistance was associated with a modest (about 2-fold) increase in the dihydrofolate reductase (DHFR) content according to radioligand-binding assay, and in the rate of cell division. In addition there was a 4-fold decrease in the fraction of long-chain MTX polyglutamates MTX(G4-6) in the cell after 24 h exposure to either 0.2 or 2.0 microM MTX. When the SCC25/CP[1] cells were kept out of CDDP for 8-9 months and 12 months to give the sublines SCC25/CP[2] and SCC25/CP[3], respectively, MTX sensitivity to continuous exposure returned to normal. The SCC25/CP[3] cells still exhibited a slightly elevated DHFR level, but their generation time became shorter than that of the parental SCC25 line. In addition the SCC25/CP[3] cells had an initial uptake velocity (V0) for MTX that was 9-fold greater than the V0 of the SCC25 or SCC25/CP[1] cells, while its ability to form MTX(G4-6) was comparable to that of the SCC25 cells. When SCC25/CP[2] cells were rechallenged with weekly 0.2 mM CDDP pulses for 4-6 months, a MTX-resistant line, SCC25/CP[4], was produced. The SCC25/CP[4] cells retained a slightly elevated DHFR content and a high proliferation rate, but the V0 for MTX influx was intermediate between SCC25 and SCC25/CP[3] cells. The ability to form the longer-chain polyglutamates MTX(G4-6) was again impaired. Thus, MTX cross-resistance can develop in cultured head and neck carcinoma cells when CDDP is used as the selecting agent for primary resistance. MTX resistance is multifactorial, as it is when MTX itself is used as the selecting agent, and appears to involve various combinations of altered growth rate, DHFR content, MTX uptake, and ability to form noneffluxing long-chain MTX polyglutamate species. These results are potentially of clinical relevance, since CDDP and MTX are often used in combination with other drugs or with radiation to treat patients with squamous cell carcinoma of the head and neck.

Pharmacokinetics of actinoymcin D in patients with malignant melanoma.

The distribution and excretion of tritiated actinomycin D have been determined in 3 adult patients with disseminated malignant melanoma. In the blood, the drug was preferentially taken up into nucleated cells. The urinary and fecal excretion was prolonged and only about 30 per cent of the dose of actinomycin was recovered in 9 days. There was evidence that the drug was concentrated in bone marrow and tumor cells, but did not readily cross the blood-brain barrier. The long tissue half-lige of actinomycin D suggeststhat an intermittenr schedule of administration would be the most effective.

Actinomycin D oxazinones as improved antitumor agents.

1,4-Oxazinone derivatives of the phenoxazinone chromophore in actinomycin D (AMD) have been synthesized by condensation of AMD with alpha-keto acids. By varying the starting alpha-keto acid, the substitutions on the oxazinone ring and, consequently, the lipophilicity of the molecule could be altered. These oxazinone derivatives revert to AMD in physiological media and it appears that these oxazinones are "depot" forms of AMD and possess physicochemical and DNA-binding properties which are significantly different from those of AMD. The oxazinones, which have bulky and lipophilic substituents at position 3, demonstrate more pronounced antitumor activity against P388 mouse leukemia and are less toxic than AMD.

N2- and C-7 substituted actinomycin D analogues: synthesis, DNA-binding affinity, and biochemical and biological properties. Structure-activity relationship.

N2-n-Alkyl- and omega-amino-n-alkylactinomycin D and 7-alkoxy-, 7-aralkoxy-, and 7-(acyloxy)actinomycin D were synthesized by modification of the parent actinomycin D molecule at the N2 and C-7 positions of the phenoxazinone moiety. The intermediate for N2 substitution was 2-deamino-2-chloroactinomycin D. For C-7 substitution, 7-hydroxyactinomycin D was used as the intermediate. Treatment of 2-deamino-2-chloroactinomycin D with an excess of the appropriate amine produced the N2-substituted derivatives. Condensation of the required alkyl or acyl halides with 7-hydroxyactinomycin D, aided by solid anhydrous potassium carbonate, yielded the C-7-substituted analogues. Calf thymus DNA-binding affinity was determined by equilibrium binding and also by thermal denaturation of DNA techniques, inhibitory activity of nucleic acid synthesis was examined using P388 cells in vitro, cytotoxicity measurements to tumor cells in vitro employed human lymphoblastic leukemic cells (CCRF-CEM), and antitumor activity was assayed against P388 mouse leukemia in CDF1 mice. Synthesis of a number of new analogues in each series and determination of the biophysical, biochemical, and biological properties established a more thorough structure-activity relationship in these analogues. These results establish that with the selection of omega-(n-alkylamino) groups at the N2 site or O-n-alkyl or O-acyl groups at the C-7 site a variety of modifications can be carried out on the actinomycin molecule while preserving biological activity. N2-3'-Amino-n-propyl- and N2-10'-amino-n-decylactinomycin D, 7-methoxy- and 7-ethoxyactinomycin D, and the 7-O-(1'-adamantoyl) ester of 7-hydroxyactinomycin D were found to be the most effective antitumor agents in vivo and in vitro. They also strongly inhibit cellular RNA and DNA synthesis and, with the exception of the ester, retain high DNA-binding affinity.

Induction of a 70 kD protein associated with the selective cytotoxicity of beta-carotene in human epidermal carcinoma.

Beta-carotene and canthaxanthin at concentrations of 70 or 300 microM were shown to inhibit the proliferation of cultured human squamous cells (SK-MES lung carcinoma and SCC-25 oral carcinoma) in a 5 hr cell density assay. Responses were similar for both tumor cell lines, ranging from 71-84% inhibition. In contrast, equimolar concentrations of alpha-tocopherol gave only 19-36% inhibition of SCC-25, but 50-75% inhibition of SK-MES cell density. Equimolar reduced glutathione resulted in 4-15% stimulation of SCC-25 and 22-25% inhibition of SK-MES cell proliferation. With cultured normal keratinocytes, treated final cell densities did not differ significantly from those of controls. Two additional assays measuring the metabolic generation of formazan (MTT assay) and [5-3H]thymidine incorporation were in substantial agreement with the growth inhibition pattern. Thus both continuous and cyclic cellular processes are involved in the tumor-specific response. Onset of the response to beta-carotene alone or in combination with alpha-tocopherol is signalled within 1-2 hours of treatment by the appearance of a unique 70 kD heat-shock protein.