H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo.

We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.

Sulfonimidamide analogs of oncolytic sulfonylureas.

A series of sulfonimidamide analogs of the oncolytic diarylsulfonylureas was synthesized and evaluated for (1) in vitro cytotoxicity against CEM cells, (2) in vivo antitumor activity against subaxillary implanted 6C3HED lymphosarcoma, and (3) metabolic breakdown to the o-sulfate of p-chloroaniline. The separated enantiomers of one sulfonimidamide analog displayed very different activities in the in vivo screening model. In general, several analogs demonstrated excellent growth inhibitory activity in the 6C3HED model when dosed orally or intraperitoneally.

H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment.

The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.

Comparison of the antitumor activity of gemcitabine and ara-C in a panel of human breast, colon, lung and pancreatic xenograft models.

Gemcitabine is a new deoxycytidine analog that exhibits significant cytotoxicity against a variety of cultured murine and human tumor cells. The cytotoxic action of gemcitabine appears to be due to the inhibition of DNA synthesis by inhibition of ribonucleotide reductase and by competition with dCTP for incorporation into DNA. We have previously shown that gemcitabine, but not cytosine arabinoside (ara-C), has a broad spectrum of antitumor activity against 7 different types of murine solid tumors.

Studies on the mechanism of sulofenur and LY295501 toxicity: effect on the regulation of cytosolic calcium in relation to cytotoxicity in normal and tumorigenic rat kidney cell lines.

Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing.

Medicinal chemistry of difluoropurines.

A series of over 70 difluoropurine analogs was synthesized by varying the C-2, 6 and 8 substituents about the purine ring system. After initial in vitro and in vivo screening, testing concentrated on the 2,6-diaminopurine analog (dFdAP) and the guanosine analog (dFdG). dFDAP appears to be a prodrug for dFdG.

Characterization of the intracellular distribution and binding in human adenocarcinoma cells of N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), a photoaffinity analogue of the antitumor diarylsulfonylurea sulofenur.

A photoactivatable diarylsulfonylurea, N-(4-azidophenylsulfonyl)-N'-(4-chlorophenyl)urea (LY219703), has been examined as a potential probe to elucidate the intracellular distribution and binding of antitumor diarylsulfonylureas. Our results demonstrated that against the human colon adenocarcinoma cell line GC3/c1, LY219703 is a more potent cytotoxic agent than N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (Sulofenur; ISCU), whereas a subline selected for resistance to ISCU was cross-resistant to LY219703, suggesting a similar mechanism of action or resistance. Cellular pharmacology studies showed that [3H]LY219703 concentrated in cells, and that its concentrative accumulation could be inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that it was similar to other antitumor diarylsulfonylurea (DSU) drugs examined.

Sulofenur cytotoxicity and changes in cytosolic calcium and mitochondrial membrane potential in human colon adenocarcinoma cell lines.

Sulofenur treatment (12.5 microM-1 mM) of colon adenocarcinoma cell lines resulted in dose- and time-dependent cell killing. LYc5 cells were viable longer than GC3/c1 cells.

Effect of albumin on antitumor activity of diarylsulfonylureas.

Several diarylsulfonylureas (DSU), including Sulofenur (LY186641) and LY181984, have been described that exhibit wide spectrum and high therapeutic activity against murine solid tumors and human tumor xenografts. The mechanism for antitumor activity is poorly understood. Moreover, in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity for DSU.

SPOT: an improved differential screening protocol that allows the detection of marginally induced mRNAs.

We have developed a differential screening technique, single plate one transfer (SPOT), that allows the easy detection of mRNAs induced only 2-fold to 3-fold or less above background. As a model system, we looked at the induction of mRNA by parathyroid hormone (PTH) in ROS 17/2.8 rat osteosarcoma cells.

Effects of diarylsulfonylurea antitumor agents on the function of mitochondria isolated from rat liver and GC3/c1 cells.

Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action.

17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens.

The effect of 17 beta-estradiol on interleukin-6 (IL-6) synthesis was examined in murine bone marrow-derived stromal cell lines, normal human bone-derived cells, and nontransformed osteoblast cell lines from mice and rats. In all these cell types IL-6 production was stimulated as much as 10,000-fold in response to the combination of recombinant interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). Addition of 17 beta-estradiol in the cultures exerted a dose-dependent inhibition of IL-1-, TNF-, and IL-1 + TNF-induced production of bioassayable IL-6.

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine).

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay.

Phase I clinical and pharmacokinetic study of LY 195448.

LY 195448 is a phenethanolamine that has shown anti-tumour activity in a range of murine tumour models, although its mechanism of action is unknown. Pre-clinical studies have indicated the absence of "standard" side effects such as myelosuppression and gastrointestinal toxicity. The present phase I trial was carried out in nine patients at doses ranging up to 133 mg/m2.

Large-scale production of monoclonal antibodies in suspension culture.

Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity.

Contractility and protein phosphorylation in cardiomyocytes: effects of isoproterenol and AR-L57.

The cardiotonic drugs AR-L57 [2-(2,4-dimethoxyphenyl)-1H-imidazo(4,5b)-pyridine] and isoproterenol stimulated contractility in cultured heart cells in concentration-dependent manners; only the effects of isoproterenol were blocked by propranolol. Isoproterenol, but not AR-L57, enhanced the phosphorylation state of seven protein bands [relative molecular weights (MrS) 155,000, 96,000, 27,000, 24,000, 20,000, 16,000, 12,000] and resulted in the dephosphorylation of one protein band (Mr 21,000). Also, only isoproterenol increased the activation states of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and glycogen phosphorylase.

Molecular basis for the cardiovascular activities of amrinone and AR-L57.

It has been suggested that amrinone and AR-L57 enhance cardiac contractility either by inhibiting phosphodiesterase activity or altering Ca++ homeostasis. Because these novel agents are potentially useful in the management of heart failure, it was of interest to more clearly define their mechanism(s) of action. Amrinone and AR-L57 caused concentration-dependent increases in the contractile states of either perfused guinea-pig hearts or cultured rat cardiomyocytes.

Chlorpromazine inhibits mitosis of mammalian cells.

Chlorpromazine (CPZ) at minimally effective concentrations accumulates mammalian cells in mitosis without lethal effects on the cells. Star-metaphase morphology similar to effects seen with classical antimitotic compounds probably results from the preferential action of CPZ on a specific class of microtubules--the pole-to-pole microtubules of the mitotic spindle. At CPZ concentrations of 8 X 10(-6) M, flow cytometry indicates no effect of CPZ on the progress of cells through phases of the cell cycle other than mitosis (M).

Evidence for selective regulation of the phosphorylation of myocyte proteins by isoproterenol and prostaglandin E1.

Both isoproterenol and prostaglandin E1 increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with 32PO3-(4), 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated 32PO3-(4) incorporation into 16 proteins, including 2 phosphoproteins not observed under control conditions.

Correlation of the clinical neurotoxicity of the vinca alkaloids vincristine, vinblastine, and vindesine with their effects on cultured rat midbrain cells.

Clinical experience with three vinca alkaloids currently in use as antineoplastic agents has shown a difference in the degree of peripheral neurotoxicity manifested by these compounds: vincristine greater than vindesine greater than vinblastine. This phenomenon may reflect differences in pharmacokinetics and/or the differential response of the nerve tissue itself. Differences in pharmacokinetics can be avoided by studying the direct effects of the vinca alkaloids on primary cultures of neuronal and glial cells.

Chronotropic effect of tyramine on rat heart cells cultured with sympathetic neurons.

Dissociated newborn rat superior cervical ganglion neurons in culture without exogenous nerve growth factor survive and extend processes on a monolayer of rat heart ventricular cells. An increase in the contraction rate of the heart cells was observed in 83% of the co-cultures treated with 5 X 10(-6) M tyramine. No increase was seen in heart cell cultures without neurons.