Objective. We have recently shown that administration of long-term, lowdose methotrexate (MTX) causes severe osteopenia in female rats. This osteopenia is characterized both by decreased osteoblast function without a decrease in osteoblast numbers, and by increased bone resorption that is believed to represent a physiologic remodeling response by osteoclasts. The present study investigates the effects of varying doses of MTX on mouse bone cells in culture.Methods. Cells were obtained by sequential digestion of neonatal mouse calvariae, and cultured with fetal calf serum (10% for osteoblast-like cells and 2% for osteoclast-like cells). After 1 week, MTX was added to each culture in concentrations of 0.6 pM, 0.4 pM, 0.2 jdf, 0.1 jdf, 1 IN, and 0.5 nM. All experiments were done on 24 wells for each MTX concentration and for the controls. The effect on osteoblastic cells was assessed, at 7 days, by cell counts and by measurement of lysate alkaline phosphatase and supernatant osteocalcin levels, and, at 21 days, by analysis of the calcified matrix production, which was cultured with ascorbic acid and Fglycerophosphate. For osteoclastic cells, cell count and lysate acid phosphatase levels were determined.Resuks. Levels of osteoblastic cells and lysate alkaline phosphatase were not changed by any of the concentrations of MTX. Matrix calcification and supernatant osteocalcin levels were diminished by MTX in a dose-responsive manner. Osteoclast-like cell numbers and acid phosphatase levels were not significantly affected by MTX. Conclusion.These results suggest that diminished mouse osteoblastic cell function Occurs with very low mean concentrations of MTX, in a dose-responsive manner. The mechanism seems to be inability of the cell to synthesize and calcify matrix, possibly through defective osteocalcin production. Thus, low-dose MTX may have an important impact on bone density by slowing osteoblastic matrix production.Methotrexate (MTX) is a folate antagonist that is commonly used in the treatment of rheumatoid arthritis (RA) and other inflammatory diseases. It is known that administration of MTX in high doses to treat childhood leukemias and breast cancer has been associated with the development of bone pain, severe osteopenia, and fractures (14). The inhibition of conversion of folic acid to tetrahydrofolic acid by competitive inhibition of the enzyme, dihydrofolate reductase, and the subsequent inhibition of DNA synthesis, accounts for the antiproliferative effects of MTX on cancer cells, and may account for the osteopenia observed in these patients (1). Limited human data on high-dose regimens ( 2 4 , however, point to stimulation of bone resorption, rather than decrease in bone formation, as the etiologic mechanism of osteopenia in these patients. Our previous studies investigated the skeletal effects of long-term, low-dose MTX in rats, using a regimen similar to that used for inflammatory disease states. In those studies, we demonstrated significant osteopenia, which was characterized by diminished osteoblast fu...

A distilled water lavage is sometimes used during tumor surgery in an effort to kill tumor cells spilled into a cavity or wound. To test the efficacy of this technique, a model study utilized nine different human tumor cell lines, subjected in v i m to hypotonic exposure for I to 10 minutes. Only the carcinoid, multiple myeloma, leiomyosarcoma cell lines, and normal lymphocytes were destroyed by the treatment. Although breast, ovarian, gastric, bladder, and melanoma cell lines were damaged to varying degrees, viable cells persisted in all cases. These data suggest that hypotonic shock is not an effective method to kill human tumor cells.Cancer 55:2779-2782, 1985.OLE AND COFFEY' reported that cells released from C the tumor margins cut during surgical excision provide a potential seed population for local tumor recurrences. Seeding may be especially troublesome when en bloc excision is not possible. In an effort to destroy such cells, Welsh and Welsh2 have advocated irrigation with warm distilled water on the premise that tumor cells would be lysed by hypotonic shock. In practice, this procedure usually involves filling the affected cavity wound, or hollow organ, with sterile water for I to 2 minutes. Many surgeons continue this practice. Although this procedure may flush cells from the area, no evidence of cell killing has been reported. Controlled evaluation of this technique would seem to be appropriate to establish its efficacy. To this end, we measured the sensitivities of nine human tumor cell lines to osmotic shock in vitro. These cell lines were challenged for I to 10 minutes with distilled water and their subsequent viability and replicative potential were assessed. Materials and Methods Cell LinesNine human tumor cell lines and peripheral lymphocytes from one donor were used. The ovarian (COLO 319), breast (COLO 533N), colon carcinoid (COLO 321), and multiple myeloma (RPMI 8226) cell lines were provided by Dr. George E. Moore, Denver General Hospital. The melanoma (FAMC 113 and I15), leiomyosarcoma (FAMC 151), gastric (FAMC 187), and transitional cell of the bladder (FAMC 191) cell lines were established in this laboratory. Cell lines were screened for contamination and verified as to transformed state and tissue type by karyotype analysis and heterotransplantation. Purified lymphocytes, concentrated over Histopaque (Sigma, St. Louis), were obtained from a healthy donor. All cultures were maintained in antibiotic free GEM 17 17 supplemented with 10% fetal bovine serum. With the exception of the multiple myeloma and normal lymphocytes, all the cell lines grew attached to the plastic substrate.