Mechanisms of growth inhibition by nonsteroidal antioestrogens in human breast cancer cells

Treatment of MCF7 human mammary carcinoma cells with the nonsteroidal antioestrogens, tamoxifen and clomiphene, leads to a concentration-dependent decrease in cellular proliferation rate which can be resolved into oestrogen-reversible and oestrogen-irreversible components. This became more clearly apparent when cells were treated with the 4-hydroxylated derivatives of these compounds where, because of enhanced affinity for the oestrogen receptor (ER), the dose-response curves for the two components could be separated. Thus treatment with 4-hydroxyclomiphene resulted in a distinct biphasic effect on cell growth. In the concentration range 10(-10)-10(-8) M, cell proliferation was inhibited in a concentration-dependent manner to a maximum of 60-70%, there was no further effect between 10(-8) and 10(-6) M, but at concentrations greater than 10(-6) M there was another concentration-dependent decrease in cell growth. Studies with a series of vinyl-substituted hydroxytriphenylethylenes revealed that in the nanomolar concentration range, where the effects of the drugs could be completely negated by the simultaneous addition of oestradiol, the potency for growth inhibition was highly correlated with affinity for ER. Such data provide strong evidence that in this concentration range the growth inhibitory effects of nonsteroidal antioestrogens are mediated by the intracellular ER. In the micromolar concentration range the effects of antioestrogens are not completely reversed by oestradiol, potency is not well correlated with affinity for either ER or the antioestrogen binding site (AEBS) but the effect is cell cycle phase-specific. Furthermore, the disparity between the affinity for AEBS (0.8-3.3 nM) and the concentration of drug needed for oestrogen-irreversible growth inhibition (greater than or equal to 2.5 microM) argue against a central role for AEBS in mediating this effect. The observation that triphenylethylene antioestrogens are calmodulin antagonists may provide some insight into potential mechanisms for this oestrogen-irreversible effect. Indeed, in identical experiments two phenothiazine calmodulin antagonists inhibited MCF 7 cell proliferation at concentrations greater than or equal to 2.5 x 10(-6) M. Growth inhibition following administration of fluphenazine, perphenazine and triphenylethylene antioestrogens was accompanied by qualitatively similar changes in the cell cycle kinetic parameters, i.e. accumulation in G1 phase at the expense of S phase cells. These data suggest triphenylethylene antagonism of calmodulin activated cellular processes as a potential mechanism for the oestrogen-irreversible effects of the nonsteroidal antioestrogens.     

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.     

Medical treatment of inflammatory bowel disease: new therapies,new drugs.

Ulcerative colitis, ulcerative proctitis and Crohn''s disease are chronic inflammatory conditions that affect the gastrointestinal tract. Conventional treatment has stressed the role of anti-inflammatory agents to suppress the inflammatory response. New compounds that can deliver 5-aminosalicylic acid to the colon have recently been released in Canada. Metronidazole and azathioprine may also be of benefit in Crohn''s disease. Therapy with cyclosporine and clonidine should be based on the results of further clinical trials. The use of nutritional support as primary therapy in Crohn''s disease appears promising. At present, both pharmacologic and nutritional therapies should be considered in the treatment of inflammatory bowel disease.     

The frequency of gonadotropin-releasing hormone secretion regulates expression of alpha and luteinizing hormone beta-subunit messenger ribonucleic acids in male rats

The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate, testosterone-replaced male rats. GnRH pulses (25 ng/pulse) or saline to controls, were given via a carotid cannula at intervals of 7.5-240 min for 48 h. alpha and LH beta mRNA concentrations were 109 +/- 23 and 30 +/- 5 pg cDNA bound/100 micrograms pituitary DNA, respectively, in saline controls. GnRH pulse intervals of 15, 30, and 60 min resulted in elevated alpha and LH beta mRNAs (P less than 0.01) and maximum responses (4-fold, alpha; 3-fold, LH beta) were seen after the 30-min pulses. Acute LH release to the last GnRH pulse was seen after the 15-, 30-, and 60-min pulse intervals. In contrast, LH subunit mRNAs were not increased and acute LH release was markedly impaired after the rapid (7.5 min) or slower (120 and 240 min) pulse intervals. Equalization of total GnRH dose/48 h using the 7.5- and 240-min intervals did not increase LH subunit mRNAs to levels produced by the optimal 30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates expression of alpha and LH beta mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit mRNA concentrations are associated with continuing LH responsiveness to GnRH.     

Epidermal growth factor partially reverses the inhibitory effects of antiestrogens on T 47D human breast cancer cell growth

When T 47D human breast cancer cells were treated with 10 nM of the potent antiestrogen, 4-hydroxyclomiphene, growth rate was reduced to about 50% of control. Simultaneous treatment with epidermal growth factor (EGF) and 4-hydroxyclomiphene led to a partial reversal of the growth inhibitory effect of the antiestrogen. The effect of EGF was concentration-dependent being half-maximal at 0.10 ng/ml (0.02 nM) and maximal at concentrations greater than 0.5 ng/ml (greater than 0.08 nM). Furthermore, EGF partially reversed the growth inhibitory effects of several other antiestrogens including tamoxifen, 4-hydroxytamoxifen, and LY 117018. These results are compatible with the hypothesis that part of the growth inhibitory effects of antiestrogens on breast cancer cell proliferation are mediated by inhibition of autocrine secretion of growth stimulatory peptides acting through the EGF receptor.     

Pyridinium crosslinks of bone collagen and their location in peptides isolated from rat femur

The relative proportions of pyridinoline and deoxypyridinoline in bone showed large species variations, although the total number of pyridinium crosslinks in rat, rabbit and bovine bone collagen was only 25-30% of that found in articular cartilage. Three pyridinium-containing peptides were isolated from cyanogen bromide digests of rat femoral bone and were characterized by their Mr values and amino-acid compositions. The results showed that pyridinoline and its deoxy analogue were equally distributed at two locations stabilizing the 4D stagger through interactions involving both the N- and C-terminal telopeptide regions. Less than stoichiometric amounts of pyridinium crosslinks were present in the peptides, suggesting that the isolated peptides contained additional (unidentified) maturation products of the bifunctional, reducible crosslinks.     

The synthesis of polyamide-oligonucleotide conjugate molecules.

We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.