Cation radius effects on the helix-coil transition of DNA. Cryptates and other large cations.

Most polyelectrolyte theories of the effect of ions on the thermal melting of DNA assume that the predominant influence of the cations comes through their charge. Ion size and structure are treated, for analytic convenience, as negligible variables. We have examined the validity of this assumption by measuring the melting temperature of calf thymus DNA as a function of salt concentration with four univalent cations of different hydrated radii. These are K+ (3.3 A), (n-Pr)4N+ (4.5 A), (EtOH)4N+ (4.5 A), and C222-K+ (5 A). C222-K+ is a complex of cryptand C222 with K+. With K+ as the sole cation, Tm varies linearly with the log of ionic strength over the range 0.001-0.1 M. With all the K+ sequestered by an equimolar amount of C222, Tm is depressed by 10-20 degrees C and the slope of Tm vs. ionic strength is lower. At low ionic strength, an even greater reduction in Tm is achieved with (n-Pr)4N+; but the similar-sized (EtOH)4N+ gives a curve more similar to K+. Theoretical modeling, taking into account cation size through the Poisson-Boltzmann equation for cylindrical polyelectrolytes, predicts that larger cations should be less effective in stabilizing the double helix; but the calculated effect is less than observed experimentally. These results show that valence, cation size, and specific solvation effects are all important in determining the stability of the double-helical form of DNA.

Regulation of ornithine decarboxylase gene expression in MCF-7 breast cancer cells by antiestrogens.

Ornithine decarboxylase (ODC) is an enzyme intimately related to cell growth regulation. The metabolic products of ODC, the polyamines, are known to play a vital role in the structure and function of biological macromolecules including nucleic acids and proteins. The activity of ODC is stimulated by estrogens in their target cells. In order to gain insight into the molecular mechanism of action of antiestrogens in human breast cancer, we studied the effect of tamoxifen and 4-hydroxytamoxifen on the concentration of ODC mRNA, ODC activity, and the polyamine levels in a hormone-responsive breast cancer cell line, MCF-7. ODC mRNA concentration was reduced to 40% of the controls after 6 h of treatment of the cells with 100 nM 4-hydroxytamoxifen, but tamoxifen had no significant effect on ODC mRNA after treating with even 1 microM concentration for 36 h. ODC activity was, however, reduced to 40 and 75% of the controls after 24 h of treatment with 4-hydroxytamoxifen and tamoxifen, respectively. There was a significant reduction in the concentration of putrescine to 63% of control in tamoxifen-treated cells, but spermidine and spermine levels were not affected. With 4-hydroxytamoxifen, putrescine, spermidine, and spermine levels were reduced to 41, 62, and 79% of the control, respectively. In addition, exogenous putrescine was able to reverse the growth inhibitory effects of 4-hydroxytamoxifen. Overall, these results indicate that ODC and polyamine levels in MCF-7 cells are controlled by antiestrogens, and that suppression of polyamine biosynthesis plays a critical role in the growth inhibitory effects of antiestrogens.

Chromosomal replication asynchrony of a human breast carcinoma cell line. I. studied by continuous BrdU incorporation and G-banding analysis.

Continuous BrdU incorporation and the Giemsa staining technique were used to study the cell cycle kinetics of a human breast tumor cell line. It was found that the interchromosomal replication pattern of the neoplastic cell was significantly different from that of normal cells in two respects. First, the pattern is highly asynchronous; within a single cell there are chromosomes at different replication cycles; that is, some chromosomes complete their DNA replication before others begin. Second, the replication schedule for the chromosomes, as identified by superimposing the BrdU-Giemsa technique on the trypsin G-banding technique, is relatively consistent within the cell line but differs from that of normal cells. Some chromosomes that replicate late in normal human lymphocytes and fibroblasts replicate early in this cell line. In contrast to the unusual interchromosomal replication pattern, gross analysis of the intrachromosomal replication schedule shows no apparent difference from that reported for normal cells. The asynchrony phenomenon reported here may be associated with the etiology of aneuploidy in neoplasia.

Nonrandom abnormalities in chromosome 1 in human testicular cancers.

Trypsin G banding was performed on metaphase chromosomes from 14 cell lines derived from primary tumors or metastases of 11 patients with testicular cancer. Most of the cell lines, 11 of 14, have a modal number between 51 and 61. All lines have numerical and structural changes involving chromosome 1 with trisomy of the q arm being the common aberration. Break points in chromosome 1 were nonrandom, being concentrated in the regions of p12, q12, p36, and p22, which resulted in morphologically identical marker chromosomes in different cases. These changes probably are not artifacts of cell culture. In one instance, three lines derived from the same patient, one from tissue removed at operation, and two from separate metastases removed at autopsy nearly 3 years later after unsuccessful radiotherapy and chemotherapy had identical chromosome compositions. In another case, lines derived from a primary tumor and a metastasis from the same patient also had identical marker chromosomes. The consistent involvement of chromosome 1 in aberrations may be associated with the highly malignant nature of testicular cancers.

Abnormal ornithine carbamoyltransferase in mice having the sparse-fur mutation.

Mice with the X-chromosomal sparse-fur (spf) mutation frequently have urinary bladder stones composed mostly of orotic acid, which was identified by the following criteria: ultraviolet and infrared absorption, spectra, chromatographic behavior, melting point, and reactivity in a specific color test. This clue led to the discovery that spf-bearing mice have an abnormal form of liver ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3). Normal ornithine carbamoyltransferase has maximum activity at pH 7.6-8.0 and 80% of maximum activity at pH 10.0.