Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin

Three sublines of NIH 3T3 cells had the properties of non-neoplastic, preneoplastic, and neoplastic cells, respectively. The closer the cells were to neoplastic behavior, characterized by continuing growth at high density, the slower they multiplied at lower density. Under the conditions of high population density and low calf serum concentration used in the assay for transformed focus formation, the transformed or neoplastic cells were much more sensitive to killing by methotrexate (MTX) than were non-neoplastic cells in the same culture. This differential sensitivity of neoplastic cells was far more pronounced in molecular, cellular, and developmental biology medium 402 (MCDB 402) than in DMEM. It is associated with the presence in MCDB 402 of folinic acid, known clinically as leucovorin, which is a reduced form of the folic acid present in DMEM. Although leucovorin had been shown to selectively spare normal bone marrow and intestine in animals from the killing effect of MTX on tumor cells, we demonstrate the preferential killing of neoplastic over non-neoplastic cells of the same derivation. Neither neoplastic nor non-neoplastic cells were killed once they had stopped multiplying at their respective saturation densities. The development of the light foci characteristic of the preneoplastic cells was less sensitive to MTX than the formation of the dense foci produced by the fully neoplastic cells. The system should serve as a valuable model to establish basic principles and optimal conditions for selective killing of neoplastic cells by chemotherapeutic drugs.

Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20–50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9–12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines.

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.