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We report a functional link between expression of the metastasis suppressor gene nm23 and cancer cell sensitivity to the alkylating agent cisplatin. Cisplatin was 2-15-fold more inhibitory to the growth in vitro of nm23 transfectants of the K-1735 TK murine melanoma, MDA-MB-435 human breast carcinoma, and OVCAR-3 human ovarian carcinoma cell lines as compared to matched control transfectants. Administration of a single dose of cisplatin i.v. after injection of control- or nm23-1-transfected K-1735 TK melanoma cells resulted in a more pronounced inhibition of pulmonary metastatic colonization by the nm23-1 transfectants. The mechanism of nm23-dependent sensitivity to cisplatin is unknown, but was correlated with increased formation of interstrand DNA cross-links in nm23-H1 transfected breast carcinoma cells. These data suggest that elevation of tumor cell nm23 expression may be considered as a potential therapeutic strategy in combination with cisplatin treatment.

We report the phenotypic effects of transfection of human nm23-H1 cDNA into the human MDA-MB-435 breast carcinoma cell line. Upon mammary fat pad or subcutaneous injection into nude mice, both the nm23-H1 and control transfected lines produced primary tumors; however, the nm23-H1-transfected lines produced metastases in significantly fewer mice than did control transfected lines. Reductions in tumor metastatic potential in vivo were accompanied by decreased colonization in soft agar and an altered colonization response to transforming growth factor beta in vitro. Total nucleoside diphosphate kinase activity, an enzymatic activity possessed by the Nm23 family, was not directly correlated with Nm23-H1 expression levels or suppression of metastatic potential in all cases examined. The data establish that nm23-H1 has functional suppressive effects on the tumor metastatic potential of a human breast carcinoma cell line, and suggest that it may regulate signal responsiveness in the colonization response.

The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning the nm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases in nm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which human nm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate that nm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.

Reduced expression of the nm23 gene in certain rodent model systems and human breast tumors has been correlated with high tumor metastatic potential. To investigate the functional effects of nm23 expression, we have transfected a constitutive murine nm23-1 expression construct into highly metastatic K-1735 TK murine melanoma cells. TK clones expressing the exogenous nm23-1 construct exhibited a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential independent of tumor cell growth, and altered responses to the cytokine transforming growth factor beta 1 in soft agar colonization assays, compared with control-transfected TK clones. In contrast, nm23-1-transfected TK clones exhibited no significant differences in intrinsic tumor cell growth, i.e., primary tumor size in vivo, anchorage-dependent growth rate in vitro, and anchorage-independent colony formation in soft agar in vitro. The data demonstrate a suppressive effect of nm23 on several aspects of the cancer process, including tumor metastasis.

Two sublines of the T24 human urinary bladder carcinoma cell line which differ in tumorigenicity in nude mice have been studied (T24A and T24P). T24A obtained directly from the American Type Culture Collection is non-tumorigenic while T24P obtained after multiple passages in several NCI laboratories produces tumors in 100% of inoculated mice. T24P cells differ morphologically from T24A, have a higher saturation density, are less serum-dependent for growth, and are more sensitive to ouabain toxicity. Cytogenetic studies show that the 2 sublines differ significantly in chromosome number, with a modal chromosome range of 76-89 in T24A and a modal chromosome number of 48-51 in T24P. Southern blot analysis of MspI cleaved T24A and T24P DNAs with the H-ras SmaI probe indicates that both contain only the activated mutant allele originally described in T24. Northern blot analysis shows equal amounts of the 1.2kB ras polyadenylated message, and immunoblotting with rasHa antibody demonstrates no significant difference in the amounts of ras proteins. These results indicate that 2 sublines of a ras oncogene-containing tumor cell line can differ greatly in tumorigenicity and other in vitro characteristics of transformation, and yet have similar expression of the ras oncogene. The fact that the tumorigenic cell line contains fewer chromosomes suggests that tumorigenicity may be related to the loss of some regulatory gene.

We have adapted T24P, a tumorigenic subline of the T24 human bladder cancer cell line, to grow in 5 mM butyrate. In the presence of butyrate, the adapted cells (T24P/B) grow more slowly than the unadapted cells (T24P/C), have a lower saturation density, increased serum requirement for growth, loss of ability to form colonies when plated at low cell density, and decreased ouabain sensitivity. Morphologically, T24P/B cells in butyrate are large and flattened with increased cytoplasm. When T24P/B cells are grown without butyrate, the morphological changes, growth rate, plating efficiency, and ouabain sensitivity return to those of T24P/C. While the saturation density increases, it does not return to levels of T24P/C, and the size of colonies never reaches that of the T24P/C colonies. Both T24P/C and T24P/B are tumorigenic in nude mice, however, the T24P/B tumors differ grossly and microscopically from those produced by T24P/C in that they contain large cystic structures filled with clear fluid and lined by transitional cell epithelium with flattened surface layers. Although the transformed phenotype and tumorigenicity of T24P are modified by adaptation to growth in butyrate, no significant changes in ras oncogene RNA or protein expression were identified.

Since butyrate inhibits DNA synthesis and cell growth, we adapted NIH 3T3 cells to grow in butyrate to study its effects on retroviral cell transformation. In cultures of butyrate-adapted NIH 3T3 cells infected with Harvey Sarcoma Virus, no foci were formed; however, there was evidence of retroviral replication, and the P21 oncogene product was demonstrated by immunofluorescence in its characteristic localization beneath the cytoplasmic membrane of single cells and pairs of cells. When the butyrate was removed, these cells proliferated to form foci. Butyrate therefore does not prevent oncogene expression but does reversibly inhibit focus formation.

Both murine and human cell lines transformed by the v-Ki-ras gene have been shown to be much more sensitive to the toxic effects of the cardiac glycoside ouabain than their respective controls. This differential toxicity has previously been used in the isolation of flat revertant clones from populations of Kirsten murine sarcoma virus transformed NIH/3T3 cells. Here, we have undertaken a further characterization of this phenomenon in murine and human tumor cells. Two different techniques, a 51Cr-release assay and a quantitative Crystal violet elution assay, have been employed to compare the sensitivities to ouabain of normal and v-Ki-ras-transformed NIH/3T3 cells. In each assay, ras-transformed NIH/3T3 cell lines displayed an increased sensitivity to ouabain as compared to the parental NIH/3T3 cell line, both in dose-response and in time-course experiments. In a separate study, ouabain was also able to inhibit the growth in semi-solid medium of 2 v-Ki-ras-transformed NIH/3T3 cell lines (DT and K-NIH) in a dose-dependent fashion. The same concentrations of ouabain were effective in both the 51Cr-release and Crystal violet assays. To address the question of whether increased sensitivity to ouabain is a specific result of transformation with the ras oncogene or is a common event which accompanies transformation by other oncogenes, we have screened a variety of transformed NIH/3T3 derivatives. All of these lines displayed an increased sensitivity to ouabain when compared to the parental NIH/3T3 cell line.