George B. Boder

Mineralization and metabolic response in serially passaged adult rat bone cells.

SummaryCell populations derived from adult rat bone were grown in cell culture and characterized with respect to their morphology and response to hormones. The cells were isolated from adult rat calvaria by mechanical rather than enzymatic methods. Cultures were initiated in modified BGJb medium supplemented with fetal bovine serum. These cultures and several cloned populations derived from them retained the ability to mineralize in vitro even after extended serial passage.Cultures derived from an osteoblast-enriched population showed an initial positive cAMP response to PTH and PGE2, but not to TCT. The PTH and PGE2 responses diminished with serial passage. The PTH response was no longer measurable at passage 6, and the PGE2 response was not evident in passage 11. In one clone, the PGE2 response persisted through passage 16. Adult rat skin fibroblasts cultured similarly did not respond to PTH or TCT, but still had a significant PGE2 response through passage 21.The cultured cells formed multiple layers with localized areas of higher cell density. Mineral plaques with major diameters as great as 0.75 mm were evident in the areas of greater cell density. Less extensive mineral deposits were present throughout the culture. The mineral plaques consisted of apatite-like crystals deposited on an organic matrix. Matrix vesicles and mineralized spherules appeared to be associated with initial mineral deposition. The spherules apparently coalesced to form more complex mineralized structures. A limited amount of mineralization also was observed in rat skin fibroblast cultures.

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

SummaryGemcitabine 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 × 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.

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.

Comparative effects of some cardioactive agents on automaticity of cultured heart cells.

Abstract Cells from mouse hearts, disaggregated with collagenase were cultured in F 12 medium. These cells were viable for approximately 6 months. Using a sensitive monitoring system that allowed recording of both chronotropic and inotropic responses, we studied the effects of several cardioactive agents on the contractility of the cultured cells. The ventricular cells responded to norepinephrine, but not to guanethidine or tyramine. Since both guanethidine and tyramine release norepinephrine from adrenergic nerve endings, the lack of response to guanethidine and tyramine indicate that the heart cells are not innervated by adrenergic nerves. Atrial cells, but not ventricular cells, are responsible to acetylcholine. Dibutyryl cyclic AMP, but not cyclic AMP, elicited a positive inotropic and chronotropic response. Both positive inotropic and chronotropic responses were observed following glucagon administration and these effects were not antagonized by propanolol. Both epinephrine and T 3 elicited a positive inotropic effect but through different mechanisms. Arrhythmia and fibrillation of the cells could be induced by aconitine. The culture method and significance of responsiveness of the cultured cells are discussed.

Pharmacological properties of a marine natural product, stypoldione, obtained from the brown alga Stypopodium zonale

Stypoldione (Fig. 1), an ortho-quinone monoalcohol with a molecular weight of 426, is derived from the brown seaweed Stypopodium zonale . It was found as part of a study by Gerwick & Fenical (1981) in which mechanisms of chemical defense against foraging in the marine environment were being elucidated . Living samples of the alga had been observed to be toxic to fish in aquaria, and algal extracts were then investigated using icthyotoxicity as an indication of biological activity . Stypoldione was not the most potent of the compounds extracted from S. zona/e, but due to its chemical stability, was chosen as representative of the compounds isolated .

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.

Biological activities of 3-isoadenosine

Abstract An isomer of adenosine, 3-β- d -ribofuranosyladenine (3-isoadenosine), has been studied in a number of bacterial and mammalian cell systems. While 3-isoadenosine readily supported the growth of the adenine-requiring Escherichia coli B97, it failed to support the growth in tissue culture of a murine cell line rendered dependent on an exogenous purine source by the folic acid antagonist amethopterin. 3-Isoadenosine inhibited the growth of various mammalian cell lines in Eagles medium at levels of 10−4 to 10−6 M and it displayed a cytotoxicity for the lymphoblastic leukemia cell line L5178Y of the same order as that of 6-azauridine. When tested by an agar overlay method, 3-isoadenosine also inhibited the growth of Adeno III virus in tissue culture. In order to investigate the inhibitory activity of 3-isoadenosine, comparative experiments were carried out in the above systems with other nucleoside analogs (psicofuranine, pseudouridine, triacanthine, etc.), and an unsuccessful attempt was made to reverse this inhibition with nucleosides and other complex materials. A daily dose of 3 mg/kg given intraperitoneally to mice for ten days was well tolerated but 6.0 mg/kg was toxic. The final phase of this study was the evaluation of 3-isoadenosine in tumor-bearing and virus-infected animals. The interpretation of the observed biological activity in terms of the underlying biochemical mechanisms has been attempted by comparison of the activities of 3-isoadenosine and of related nucleosides and other agents. This comparison revealed a striking similarity in the characteristics of inhibition of mammalian cells in tissue culture by 3-isoadenosine and by 7-deaza-adenosine.

Extended Production of Insulin by Isolated Rabbit Pancreatic Islets; Evidence for Biosynthesis of Insulin

Summary Methods for prolonged primary suspension cultures of rabbit pancreatic islets were established, and incorporation of carbon-14 labeled amino acids into insulin and other proteins was demonstrated.

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.

Metabolites from animal and plant cell culture.

Publisher Summary This chapter compares transformation of organic compounds in cell cultures of higher plant and animal origin. It includes reports of any metabolites that have been produced by cell cultures, with the exception of cells from insects, invertebrates, and cold-blooded vertebrates. Although animal cells presumably contain all genetic material necessary for totipotentiality, a marked difference between animal and plant cells is noted while demonstrating this phenomenon in culture. Exogenous DNA may alter synthesis of animal cellular products by induction. A large number of proteins have been produced by many types of both plant and animal cells. A number of polysaccharides and glycoproteins, as well as other high molecular weight materials, are produced in the culture of both plant and animal cells. These include the hydroxyproline-containing proteins such as collagen. The chapter closes with the future applications of plant and animal cell cultures. It states that a great deal research on animal cells are toward establishment of long-term continuous cell strains.