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/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

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. A correlative structure-activity relationship to the oncolytic sulfonylureas was not apparent.

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/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

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. The activity of gemcitabine was schedule dependent. To further characterize its activity, gemcitabine was tested against 12 human carcinoma xenografts. When given on an every 3 day x 4 schedule, the following percent inhibitions (at maximally tolerated doses [MTD]; MTD/2) in tumor growth were seen: MX-1 mammary (93%; 80%), CX-1 colon (92%; 82%), HC-1 colon (96%; 92%), GC3 colon (98%; 94%), VRC5 colon (99%; 100%), LX-1 lung (76%; 61%), CALU-6 lung (75%; 38%), NCI-H460 lung (45%; 46%), HS766T pancreatic (73%; not tested), PaCa-2 pancreatic (69%; 40%), PANC-1 pancreatic (70%; 60%), and BxPC-3 pancreatic (9%; 19%). In contrast, only the LX-1 lung carcinoma xenograft was responsive to ara-C treatment, which inhibited tumor growth by a marginal 62 percent. Thus, like its activity against murine solid tumors, gemcitabine has excellent antitumor activity against a broad spectrum of human 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. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.

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. Both compounds significantly inhibited mammary tumor growth in mice, caused a moderate inhibition in ovarian and lymphosarcoma models, and demonstrated no activity in lung and melanoma models. This is a narrower spectrum of activity than that of gemcitabine (dFdC). The antitumor activity of dFdAP in human xenografts that are refractory to standard clinical agents was comparable or superior to that of gemcitabine. However, during the preliminary toxicology testing, dFdG was associated with several deaths caused by cardiac toxicity. Therefore, although dFdG is a potentially useful oncolytic, further investigation is required.

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. Accumulation of [3H]LY219703 in cells was progressively decreased by co-incubation with increasing concentrations of ISCU, and in cells incubated to steady state with 1 microM [3H]LY219703, ISCU (500 microM) rapidly displaced the photoaffinity analogue. Photoactivation of [3H]LY219703 by UV light (5-30 min) prevented efflux of radiolabeled drug during a 20-min wash in drug-free medium. Subsequent distribution studies showed that 89% of the radiolabel was associated with particulate components, and that approximately 20% of the radiolabel in the 320,000 g pellet could be extracted with acetone. Subcellular distribution showed approximately 6% associated with nuclei, 52% with mitochondria and 26% in the microsomal fraction. The effect of UV photoactivation on the distribution of [3H]LY219703 in soluble and particulate fractions was also examined in GC3/c1 cell preparations sonicated prior to being incubated with [3H]LY219703. A high proportion (83%) of radiolabel associated with the 100,000 g pellet, and distribution between soluble and particulate fractions was not altered by UV irradiation. Specific activities of protein in the 100,000 g supernatant and pellet were 0.186 and 0.537 nmol/mg, respectively. Putative binding species were analyzed by SDS-PAGE. Using SDS-PAGE, ten major binding proteins were identified in 320,000 g pellets from GC3/c1 cells: M(r) 110, 88, 76, 70, 64, 58, 48, 36, 26, and 24 kDa, and at least four of these (88, 70, 64, and 36 kDa) were also detected in mitochondria isolated from cells after photoactivation, or in mitochondrial preparations that were incubated with [3H]LY219703 and photoactivated after isolation from cells. Results suggested that under conditions of SDS-PAGE some dissociation of radiolabel from proteins also occurred. Binding of [3H]LY219703 to a model substrate, bovine serum albumin, and the effect of denaturing conditions used for sample preparation prior to SDS-PAGE, showed that relatively mild denaturing conditions (23 degrees, 2 hr) caused significant dissociation of radiolabel from BSA.(ABSTRACT TRUNCATED AT 400 WORDS)

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. Each concentration resulted in elevation of cytosolic calcium [Ca2+]i) for both cell lines. At lower doses, elevation was delayed for LYc5 cells. GC3/c1 cells after 1 mM treatment in Ca(2+)-free HBSS showed no rise of [Ca2+]i. GC3/c1 cells after carbonyl cyanide-m-chlorophenylhydrazone rapidly lost rhodamine 123 fluorescence from mitochondria; after 1 mM sulofenur, fluorescence faded slowly. Following treatment, cells became rounded, blebs formed and the cells died. Results suggest that elevated [Ca2+]i plays an important role in sulofenur cytotoxicity.

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. Since DSU are extensively bound to serum albumin (> 99%), we sought to determine the effect of albumin on tumor cytotoxicity. We adapted human CCRF-CEM leukemia and GC3 colon carcinoma cells for growth in UltraCHO serum- and albumin-free medium. In comparisons between normal growth medium (RPMI-1640 with 10% fetal bovine serum) and UltraCHO medium, the unbound fraction of drug correlated better with cytotoxic activity than did the total drug. Tumor cytotoxicity by DSU required > 24 h and was markedly enhanced in UltraCHO medium. For example, LY181984 and Sulofenur had IC50 values of 7.4 and 12.1 micrograms/ml against CCRF-CEM in normal growth medium and 0.6 and 0.2 microgram/ml in UltraCHO. Moreover, DSU with the lowest IC-50s in albumin-free medium displayed the most potent in vivo antitumor activity in the 6C3HED lymphosarcoma. A Sulofenur-resistant CCRF-CEM cell line was developed by culturing the cells for > 20 passages in UltraCHO medium containing LY186641 at 2 micrograms/ml (10X IC-50). This line showed approximately 18-fold resistance to LY186641, but did not show cross-resistance to vinblastine, actinomycin D, or doxorubicin. The albumin-free conditions may be useful for further mechanistic studies on the antitumor action by DSU. Further studies are underway to determine whether DSU structural requirements for cytotoxicity an albumin binding are intrinsically linked.

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. The basis for this technique is to symmetrically spot in quadruplicate, on a single plate, a large number of potentially positive plaques obtained from a primary, conventional screen. We then do only one transfer from this plate in order that there will be minimal variability in DNA transfer. This filter is cut into symmetrical strips so that all clones are multiply represented on each strip. These strips are then hybridized with different probes. Since each strip contains an approximately identical amount of DNA per plaque, it is possible to accurately detect mRNAs that are induced only slightly above background. Additionally, the large sizes of the DNA plaques, as well as spotting each clone serially, contribute to the sensitivity of the technique.

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. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pKa values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.

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. Testosterone and progesterone (but not 17 alpha-estradiol) also inhibited IL-6, but their effective concentrations were two orders of magnitude higher than 17 beta-estradiol. 17 beta-estradiol also decreased the levels of the IL-6 mRNA. In addition, estradiol inhibited both TNF-induced IL-6 production and osteoclast development in primary bone cell cultures derived from neonatal murine calvaria. The TNF-stimulated osteoclast development was also suppressed by a neutralizing monoclonal anti-IL-6 antibody. This in vitro evidence suggests, for the first time, a mechanistic paradigm by which estrogens might exert at least part of their antiresorptive influence on the skeleton.

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. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.

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. The major toxicities up to that dose were mild, reversible hypotension, tachycardia and tremor. No haematological or biochemical toxicity was observed. Murine pharmacokinetics were assessed at a dose level that was effective in experimental tumours and compared with human pharmacokinetic parameters derived from this study. The results indicated the clinical possibility of reaching peak drug levels associated with experimental activity. However, no responses were seen at the doses used. This study was terminated prior to its completion due to an unexplained loss of activity against murine tumours since September 1987. No significant loss of the in vitro anti-mitotic activity originally reported by Boder et al. [3] was observed. Possible reasons for the apparent loss of in vivo activity have been intensively investigated, but no cause has been determined. Therefore, clinical trials with LY 195448 have been discontinued.

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. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

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. The eight protein bands resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and detected by autoradiography were altered by isoproterenol in time- and concentration-dependent manners. The 24,000-Mr protein substrate phosphorylated in response to isoproterenol was converted to a 12,000-Mr species by heating in the presence of SDS prior to electrophoresis, suggesting that the two substrates were in fact identical proteins. A comparison of the 2-min responses to varying concentrations of isoproterenol resulted in excellent correlations between the phosphorylation states of individual protein bands and contractility. This was true even for the 21,000-Mr species that was dephosphorylated. However, only the 27,000-, 24-12,000-, and 16,000-Mr substrates were phosphorylated rapidly enough to be associated with the onset of the inotropic response. Cultured myocytes are an important feature of these studies as they are 84% pure ventricular cells that remain 100% viable throughout an experiment. Because this system is suitable for biochemical measurements and the effects of agents on heart cell contractility can be determined, it is possible to correlate changes in biochemical parameters with alterations in physiological state.

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. To determine whether these actions might result from an increase in sarcolemmal Ca++ movement, the effects of these agents on Ca++ accumulation were studied in a simple system, dog erythrocytes. Both agents promoted erythrocyte Ca++ accumulation in time and concentration-dependent manners, effects that resulted primarily from increased Ca++ entry. However, because these effects were not measurable at inotropic drug concentrations and were apparent only after a 30-min incubation, they did not provide an explanation for the inotropic effects of these agents. Amrinone and AR-L57 inhibited dog heart phosphodiesterase activity (isozyme III) with EC50 values of 23 and 420 microM, respectively; however, only the inotropic responses to amrinone were attenuated by the muscarinic agonist, carbachol, thereby implying a cAMP (cyclic AMP)-dependent mechanism. In cultured ventricular cells, concentrations of amrinone (2 X 10(-4) M) and AR-L57 (3 X 10(-5) M) that caused maximal inotropic responses were associated with the activation of glycogen phosphorylase, but neither drug significantly increased the activation state of cAMP-dependent protein kinase. To further probe the effects of these drugs on intracellular cAMP and Ca++ metabolism, their effects on protein phosphorylation were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

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). These results suggest a possible mechanism for toxic side effects of CPZ in man such as granulocytopenia and light sensitization.