We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.
Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.
Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11- bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 µM. Another group of three compounds showed antiproliferative activity at about 5 µM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.Key words: polyamine analogs, breast cancer cells, apoptosis, molecular modelling.
Estrogens stimulate the growth of a majority of estrogen receptor (ER)-positive breast cancer cells. In contrast, estradiol exerted a 75% inhibition of DNA synthesis in the MCF-10AE(wt5) cell line, obtained by the transfection of the ER gene into a normal breast epithelial cell line, MCF-10A. The estradiol-mediated growth inhibitory effect was reversed by ICI 164384, a pure anti-estrogen. Analysis of cell cycle by flow cytometry showed a significant increase of G1 cells by estradiol treatment compared to controls. To understand the mechanism of action of estradiol on MCF-10AE(wt5) cells, we examined the level of a cyclin dependent kinase inhibitor (CKI), p21, by Western blot analysis. Our results showed a 5- to 10-fold increase in the level of p21 in estradiol-treated MCF-10AE(wt5) cells compared to controls. ICI 164384 reversed estradiol-mediated induction of p21. Northern blot analysis of p21 mRNA indicated that estradiol stimulated its message in MCF-10AE(wt5) cells. Analysis of a panel of 6 breast cancer cell lines showed the absence of p21 protein, whereas it was present at a very low level in MCF-10A cells. Comparison of p21 in MCF-10A and MCF-10AE(wt5) cells showed an abundance of p21 in the ER-transfected cells. However, this p21 appears to be inactive in the absence of estradiol. These results suggest a p21-mediated pathway as a possible mechanism for the growth inhibitory effects of estradiol on at least a subset of ER-transfected cell lines.
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