HTC Cells Research Articles

New steroidal anti-inflammatory antedrugs: Methyl 3,20-dioxo-9α-fluoro-11β,17α,21-trihydroxy-1,4-pregnadiene-16α-carboxylate and methyl 21-acetyloxy-3,20-dioxo-11β,17α-dihydroxy-9α-fluoro-1,4-pregnadiene-16α-carboxylate

Focused efforts have been made to increase local-to-systemic activity ratios of potent anti-inflammatory steroids for local and/or topical applications. The approach taken in the present investigation is based upon the concept of “antedrug,” defined as a locally active compound that exerts its action at the application site but rapidly undergoes a predictable biotransformation to an inactive metabolite that is readily excreted upon entry into the systemic circulation. In continuing efforts to synthesize potent, anti-inflammatory steroids without systemic glucocorticoid activities, 9α-fluoro-methyl11β,17α,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16α-carboxylate (FP16CM) and its 21-acetate derivative (FP16CMAc) have been synthesized and screened. Novel antedrugs were evaluated for antiinflammatory activity in the acute croton oil-induced ear edema bioassay, adverse systemic effects in the 5-day croton oil model, receptor binding, and concomitant L-tyrosine-2-oxoglutarate aminotrans-ferase (EC 2.6.1.5) (TAT) enzyme induction in HTC cells in culture. Following a single topical application in the croton oil-induced ear edema bioassay, treatment with all compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID 50 values (nmol resulting in a 50% reduction of edema) were calculated: 817, 540, 266, and 67 for hydrocortisone (HC), prednisolone (P), FP16CM, and FP16CMAc, respectively. Calculated relative potencies, setting HC = 1.0, were P, 1.5; FP16CM, 3.1, and FP16CMAc, 12.2 Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast to the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. Relative binding potencies for cytosolic HTC glucocorticoid receptors were 1.0, 20.1, 5.4, and 2.5 for HC, P, FP16CM, and FP16CMAc, respectively. As predicted by the antedrug concept, FP16CM and FP16CMAc were very weak agonists for induction of TAT in HTC cells. Collectively, results of these investigations suggest that modifications of P, which included addition of the 9-fluoro and 16-methoxycarbonyl group alone or in conjunction with a 21-acetoxy moiety, increase topical anti-inflammatory activity without significant adverse systemic effects. These new antedrugs may be useful as anti-inflammatory steroids for local applications.

Lead may affect glucocorticoid signal transduction in cultured hepatoma cells through inhibition of protein kinase C

Specific cellular sites of lead action have not been completely defined. To elucidate the effects of lead exposure on glucocorticoid-mediated signal transduction in hepatic hormonal target tissues, the induction of tyrosine aminotransferase (TAT) specific activity in the H4-IIE-C3 hepatoma cell culture model system was employed. It had been found that lead acetate (3–10 μM) exposure of HTC cells significantly reduced TAT specific activity in a concentration- and time-dependent manner. Two possible molecular targets of the lead-induced effect were investigated: interference with calcium-mediated cellular processes and calcium- and phospholipid-dependent protein kinase C (PKC) activity and isoform-type interactions. Lead acetate treatment (5 μM) reduced TAT specific activity below sodium acetate treated controls by 31%. One-half of the TAT specific activity was recovered by co-treatment with 5 μM lead acetate and 10 mM calcium chloride. As the concentration of lead acetate was increased to 10 μM, interference in calcium-mediated events also increased. Potentiation of glucocorticoid induction by phorbol myristate acetate (PMA) (300 nM) in control cells was 34%, but was abolished by exposure of cells to 10 μM lead acetate (48 h). Treatment with the kinase inhibitor genistein decreased TAT specific activity by 55% and 45% in control and lead acetate exposed cells, respectively. Following treatment with dexamethasone (100 nM), significant increases in both cytosolic and particulate PKC were noted in control cells but not lead acetate exposed cells. Western blot results indicated that lead exposure may increase PKC β and decrease PKC α translocation from cytosolic to particulate fractions, respectively. Taken together, these results suggest that glucocorticoid signal transduction pathways in HTC cells involve calcium-mediated cellular events and PKC isoforms. Exposure of cells to lead results in interference with calcium-mediated events and aberrant modulation of PKC activities. Within hormonal target cells, these may be toxic molecular sites of action of the heavy metal lead and may contribute to the overall toxicity of lead exposure.

Comparative Evaluation of Cytotoxicity and Metabolism of Four Aldehydes in Two Hepatoma Cell Lines

ABSTRACTThe metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells.The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate.The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line, with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA.Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the prescreening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.

Dominant Suppression of Lymphocyte Apoptosis by Hepatoma Cells

Suppression of apoptosis appears to contribute to the development of various diseases, including autoimmune disorders and cancer. Numerous genes that encode activators and suppressors of apoptosis have been identified; however, such genes have not been shown to be expressed in all cell types. Furthermore, the sensitivity of different cell types to induction of apoptosis varies widely. We have employed a genetic approach using somatic cell hybridization to determine if apoptosis is a dominant or a recessive process in cells. These studies have utilized cell fusion partners with differing sensitivity to induction of apoptosis. The apoptosis-sensitive cells chosen were BW5147 murine thymoma cells. These cells readily undergo apoptosis in response to glucocorticoids and calcium ionophore. The resistant fusion partners were HTC rat hepatoma cells, which possess an intact glucocorticoid signal transduction pathway but are resistant to induction of apoptosis by either agent. Neither cell type expresses detectable Bcl-2 protein. Heterokaryons were identified by their retention of fluorescent cytosolic dyes and by nuclear morphology and cell size. The three types of heterokaryons observed were intratypic HTC/HTC and BW5147/BW5147 heterokaryons and intertypic BW5147/HTC heterokaryons. Glucocorticoid receptor was shown by immunohistochemistry to undergo hormone-dependent translocation to all nuclei in intertypic heterokaryons. BW5147/BW5147 heterokaryons die after treatment with glucocorticoid and calcium ionophore, whereas both HTC/HTC and BW5147/HTC hybrids survive. The presence of multiple BW5147 cells fused to a single HTC cell did not affect this outcome. This demonstrates that HTC cells are able to dominantly suppress apoptosis in all BW5147/HTC heterokaryons. Thus, HTC cells contain activities that can suppress apoptosis in lymphocytes.

Immortalized hepatocytes as in vitro model systems for toxicity testing: the comparative toxicity of menadione in immortalized cells, primary cultures of hepatocytes and HTC hepatoma cells

Immortalized differentiated liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be valuable for in vitro toxicity testing in the pharmaceutical, chemical, food and cosmetics industries. Immortalized rat hepatocyte cell lines have been produced by transfection of SV40 DNA by electroporation or calcium phosphate precipitation. Their utility has been assessed by studying the toxicity of a model compound, menadione, and by measuring the activities of DT-diaphorase and NADPH cytochrome c reductase. Enzyme activities and toxicity were compared in freshly isolated hepatocytes, the immortalized cell lines and dedifferentiated HTC hepatoma cells. In HTC cells DT-diaphorase activity was 100-fold elevated compared with freshly isolated hepatocytes. In only one cell line, C2.1.2. (produced by calcium phosphate precipitation), was DT-diaphorase activity increased (twofold) compared with freshly isolated hepatocytes. Menadione caused loss of viability at similar concentrations (40–80 μm) in the immortalized cell lines and 24-hr primary cultures of hepatocytes, whereas HTC cells showed loss of viability only with menadione concentrations above 200 μm. The immortalized lines therefore appear to have potential for predicting toxicity and for menadione this can be correlated with the expression of DT-diaphorase.

Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs.

Selection of HTC cells in drugs that inhibit ornithine decarboxylase (ODC) has produced two cell lines, HMOA and DH23A/b, that contain increased amounts of more stable ODC. In addition to alterations in ODC, these cells appear to produce modified eukaryotic initiation factor 5A (eIF-5A) at different rates, a reaction that both requires spermidine and is essential for proliferation. Alterations to the modification of eIF-5A by spermidine cannot be accounted for by changes in eIF-5A protein or modified eIF-5A turnover. Deoxyhypusine synthetase activity is similar in the parental and variant cell lines and is unaltered by growth into plateau phase or by spermidine depletion. The increased rate of eIF-5A modification in DH23A/b cells is due to an increased accumulation of the unmodified eIF-5A precursor. Increased precursor accumulation is not due to increased eIF-5A transcription, but rather it can be attributed to a metabolic accumulation caused by growth under conditions of chronically limiting spermidine. Selection using drugs that inhibit ODC apparently does not cause alterations in the eIF-5A modification pathway. These data support the hypothesis that one of the main effects of spermidine depletion is depletion of the modified eIF-5A pool, and that this is a critical factor in the cytostasis often observed after depletion of cellular polyamines.

Thyroid Hormone Activation of Transcription Is Potentiated by Activators of cAMP-dependent Protein Kinase

We characterized the cross-talk between activators of protein kinase A (PKA) and thyroid hormone (T3) in T3 receptor (TR)-mediated transcription. U937 cells were cotransfected with a plasmid expressing the TR and a reporter plasmid containing a T3 response element (TRE) oriented either as a direct repeat or as a palindrome upstream of the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. T3 activated transcription by 10-fold. T3 response was potentiated 2.5-3-fold by activators of PKA, but an activator of protein kinase C or of guanylate kinase was ineffective. In the absence of T3, activators of PKA had no effect on transcription. TR heterodimerization with the retinoid X receptor may facilitate T3/PKA cross-talk because coexpression of the retinoid X receptor potentiated cross-talk. Synergy was not observed in JEG-3, F9, CV-1, HeLa, L929, and HTC cells, indicating that it may require cell-specific factors. Synergy required the DNA- and ligand-binding domains, but not the amino-terminal domain, indicating that T3- and TRE-induced conformational changes on the TR are essential for cross-talk. PKA phosphorylated the TR in vitro, suggesting that, like other nuclear receptors, the TR is a target for PKA. These results imply that PKA cross-talks with T3 at the level of the TRE-bound TR, enhancing its transcriptional activity in a cell-specific manner.

Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.

DH23b cells, a variant of the HTC line selected for their resistance to difluoromethylornithine, exhibit defective feedback regulation of ornithine decarboxylase (ODC) stability and polyamine transport, and accumulate ODC protein to > 1000 times normal concentrations. The components of the polyamine feedback regulation system have been examined in an attempt to understand these unusual responses. Southern-blot analysis revealed an amplification (approx. 10-fold) in ODC DNA sequence without any concomitant increase in antizyme. Moreover, the amplified ODC sequence contains a single base substitution that results in the conversion of Cys-441 into Trp. This modification has previously been shown to cause ODC stability in HMOA cells. Although antizyme activity has not been noted in DH23b cells, Western-blot analysis revealed the accumulation of antizyme protein to > 50 times that induced in parental HTC cells. This increase is consistent with a 6-9-fold increase in the half-life of antizyme in these cells, a consequence of the inability of the mutant ODC-antizyme complex to be degraded by 26 S proteasome. Associated with the stabilization of antizyme in both DH23b and HMOA cells is the appearance of two additional forms of antizyme protein with apparent molecular masses of 22 and 18.5 kDa. It is suggested that these result from proteolytic removal of discrete fragments from the N-terminal end of antizyme, perhaps an indication of an initial step in rapid antizyme turnover.

Determination of Covalently Bound Hypusine and Deoxyhypusine to Protein Using Submilligram of Protein Samples by HPLC.

A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of N epsilon-(5-aminopentyl)lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

The acute effect of lead acetate on glucocorticoid regulation of tyrosine aminotransferase in hepatoma cells

Specific cellular sites of action of the environmental pollutant, lead, have not been completely defined. The present investigations were conducted to test the hypothesis that lead exposure perturbs glucocorticoid-mediated effects in hormonal target tissues. The cell culture model chosen for these investigations was the effects of lead on glucocorticoid-regulated tyrosine aminotransferase (TAT) specific activity in the H4-II-C3 hepatoma cells. Cells were treated with 300 nM-10 μM lead acetate for 24 or 48 h in absence or presence of the inducing agent, dexamethasone. Lead dose-dependently inhibited TAT specific activity up to 52% and 61% following 24 and 48 h lead treatments, respectively. These treatment times and concentrations of lead acetate did not significantly alter total cell numbers, [ 3H]thymidine incorporation or trypan blue exclusion. Glucocorticoid receptor-binding studies yielded a K d = 8.3 nM and a B max = 290 fmol/mg protein in untreated cells versus a K d = 9.2 nM and B max = 262 fmol/mg protein in cells exposed to 10 μM lead acetate for 48 h. Treatment with lead did not significantly perturb uptake of the inducing glucocorticoids or initial cytosolic receptor-binding events. To sustain induced levels of TAT, glucocorticoid must be continuously present. Following steroid withdrawal, enzyme de-induction was significantly altered in lead-treated cells. At 6 h following dexamethasone withdrawal, TAT levels had decreased to 51% of maximum in sodium acetate-treated cells. This was significantly reduced to 33% of maximum in lead acetate-treated cells. Lead treatment of HTC cells was also shown to ameliorate PMA amplification of dexamethasone-induced TAT activity. Taken together, these results suggest that acute exposure of cells to lead may inhibit processes involved in glucocorticoid-mediated enzyme induction within the hormonal target cell. Results suggest that lead may be acting to increase the turnover of TAT by actions at the transcription, translation and/or posttranslational level. Lead may also be affecting PKC-mediated phosphorylations in the glucocorticoid-TAT signal transduction system.

Specific depletion of spermidine and spermine in HTC cells treated with inhibitors of aminopropyltransferases.

The effects of a potent spermidine synthase inhibitor, trans-4-methylcyclohexylamine (4MCHA), and a spermine synthase inhibitor, N-(3-aminopropyl)cyclohexylamine (APCHA), on polyamine biosynthesis and cell growth have been studied in rat hepatoma cells (HTC cells) in culture. Treatment of HTC cells with 4MCHA or APCHA caused a marked decrease of spermidine or spermine with a compensatory increase of putrescine and spermine or spermidine, respectively, in a dose-dependent manner, suggesting specific and potent inhibition of each target enzyme. When 250 microM 4MCHA or APCHA was administered to the cells for 8 days, spermidine was decreased to 2% of control culture or spermine below 1%, respectively, while total polyamine (sum of putrescine, spermidine, and spermine) remained almost unchanged during the culture. There were no significant changes in the growth rate during treatment with the inhibitors at 250 microM concentration. The results suggest that in the growth of HTC cells, putrescine and spermine can be substituted for most of the fraction of cellular spermidine, and spermidine for most of the fraction of cellular spermine. Of five enzymatic activities involved in polyamine biosynthesis and interconversion, S-adenosylmethionine decarboxylase activity increased 8-fold with 250 microM 4MCHA, and 3-fold with 250 microM APCHA during the treatment. This increase was partially due to the increase of half-life of the enzyme. Separate roles for spermidine and spermine in the biosynthesis of the enzyme protein were also suggested.

Brain and testis selective expression of the glutathione S-transferase Yb3 subunit is governed by tandem direct repeat octamer motifs in the 5'-flanking region of its gene.

To gain insight into mechanisms of cell type-specific transcription of class mu-glutathione S-transferase genes, the gene encoding the Yb3 subunit was cloned. Yb3 subunits are selectively expressed at high levels in rat brain and testis but not in liver or kidney. The Yb3 subunit gene spans over 6 kb and consists of 8 exons and 7 introns and a sequence consisting of tandem direct repeat consensus octamer DNA binding motifs separated by a 6 base pair (bp) spacer was identified in its 5'-flanking region. Gel shift assays with a 40 bp segment of DNA containing the two consensus octamer sequences, revealed the presence of specific binding proteins in nuclear extracts of rat brain, testis and C6 glioma cells. DNA binding activity was greatly reduced in liver, kidney and HTC cells. Reporter vectors carrying segments of the 5'-flanking region of the Yb3 subunit gene fused to a luciferase gene were introduced into C6 glioma cells which express high levels of Yb3 subunits, and into HTC cells which do not. The plasmids consisting of the Yb3 gene promoter up to, but not including, the octamer motifs did not support luciferase transcription in the C6 glioma cells, but larger fragments that included the octamer repeat sequences, effectively directed transcription in the C6 glioma cells. With mutated octameric sequences transcriptional activity was greatly reduced, and none of the same Yb3 constructs directed substantial luciferase transcription in the HTC cells. The results show that octamer motifs in the 5'-flanking region of the Yb3 subunit gene are functional and are the principal cis-acting elements that account for its discrete cell type-selective expression. This gene is one of the few known targets for octamer DNA binding transcription factors in brain.

Expression of the rat gamma-glutamyl transpeptidase gene from a specific promoter in the small intestine and in hepatoma cells.

In the small intestine and in HTC hepatoma cells, the gamma-glutamyl transpeptidase (GGT) single-copy gene is transcribed into a 2.5 kb and a 2.2 kb mRNA. Cloning of the GGT cDNA sequences from HTC cells demonstrates that the 2.5 kb mRNA (mRNA(IV-1)) differs from the other rat GGT transcripts by a 371-base unique leader sequence which maps in the gene as 2 separate exons upstream of the 3 promoters which have been previously characterized. We established that the transcription of these two mRNAs is initiated on a new promoter (promoter IV) and occurs in the small intestine, in the epididymis, and in some hepatoma cells. The primary transcript initiated on GGT promoter IV is then alternatively spliced into the 2.5 kb mRNA(IV-1) or the 2.2 kb mRNA(IV-2) which is shorter in its 5'-untranslated sequence. The rat GGT gene exhibits a complex transcriptional organization leading to the transcription of five mRNAs from four independent promoters in a tissue-specific manner. The expression of the GGT promoter IV in the HTC hepatoma cells as well as in the small intestine could reveal that the HTC-transformed cells originate from liver precursor cells which still have the capacity to evolve toward different lineages. Thus, the GGT promoter IV will be valuable to isolate factors involved in the differentiation and carcinogenic processes.

Synergistic induction of tissue-type plasminogen activator gene expression by glucocorticoids and cyclic nucleotides in rat HTC hepatoma cells.

We have reported previously that tissue-type plasminogen activator (tPA) gene expression is regulated by glucocorticoids and cyclic nucleotides in HTC rat hepatoma cells. Incubation of HTC cells with the synthetic glucocorticoid dexamethasone (Dex) transiently increases tPA messenger RNA accumulation 2-fold, whereas incubation with 8-bromo-cAMP (cAMP) alone results in a sustained 2-fold increase. Nuclear run-on studies indicate that these effects occur at the level of gene transcription. In combination, however, Dex and cAMP act synergistically to induce tPA messenger RNA levels 10- to 15-fold; this synergistic induction is at least in part transcriptional. We now report that this synergistic induction of tPA gene transcription requires concomitant protein synthesis. Furthermore, the action of Dex must precede that of cAMP, and the action of Dex requires ongoing protein synthesis, whereas the action of cAMP has no such requirement. To further investigate the mechanism of the synergistic induction of tPA gene transcription, we cloned the tPA promoter from an HTC genomic library. We established the start site of transcription in HTC cells by primer extension and determined the nucleotide sequence of 2.3 kilobase-pairs (kb) of the 5'-flanking region, including 1.7 kb of sequence not previously reported. A 2.3-kb segment of the rat tPA promoter has been ligated to a chloramphenicol acetyltransferase reporter gene and its hormonal regulation evaluated in transient and stable transfection studies in HTC cells. Although this promoter length is sufficient to mediate the 2-fold induction in gene expression seen with cAMP alone, it is not sufficient to recapitulate the synergistic induction of endogenous tPA gene transcription seen with Dex plus cAMP in combination. We have ruled out relief of transcriptional arrest as the mechanism of the synergistic induction. Therefore, we suggest that sequences lying outside the most proximal 2.3 kb of tPA promoter mediate the synergistic interaction of Dex and cAMP.

Iron Ion Induces Mitochondrial DNA Damage in HTC Rat Hepatoma Cell Culture-Role of Antioxidants in Mitochondrial DNA Protection from Oxidative Stresses

The mitochondrial DNA (mtDNA) is exposed to the reactive oxygen species generated in the mitochondrial electron transfer system. While mitochondrial superoxide dismicrotase (Mn-SOD) scavenges superoxide anions to prevent damage of mtDNA, excess amount of reactive oxygen generated by quinone compounds impairs the mtDNA, in which involvement of iron ion-catalyzed reactions is suggested. In this commicronication, we report that the mtDNA in HTC rat hepatoma cells was markedly damaged in the presence of either ferrous (Fe2+) or ferric (Fe3+) iron in the culture medium. The mtDNA of HTC cells was damaged in the presence of 100 μM of iron ions in the culture medium within 3 h. There was no significant difference in the potency of Fe2+ and Fe3+. Deferoxamine, a Fe3+-specific chelating reagent, blocked the iron ion-induced mtDNA damage completely. The mtDNA in rat hepatocyte primary culture was, on the other hand, not damaged by either Fe2+ or Fe3+. To understand the difference between iron ion-induced damage of mtDNA in HTC cells and in the primary hepatocytes, the Mn-SOD activity and lipid-soluble antioxidant contents were compared. The Mn-SOD activity in HTC cells was markedly lower (<0.02 U/mg cell protein) than that in rat hepatocyte primary cell cultures (1.5 U/mg cell protein). Immicronoreactive Mn-SOD content in HTC cells was also lower (21 ng/mg cell protein) than in the primary cultures (99 ng/mg cell protein). HTC cells contain microch smaller amounts (3-11% of that of normal rat hepatocytes) of α-tocopherol and coenzyme Q homologs (CoQn). These results suggest that reactive oxygen species produced by iron ion impaired mtDNA of HTC cells, in which antioxidant activity was markedly decreased.

Development of specific bioluminescent In vitro assays for selecting potential antimineralocorticoids

Efficient antimineralocorticoid selection requires a reliable, discriminating and easy assay for monitoring biological activity of not only the specific receptor, but also closely related receptors such as glucocorticoid and progestin. These related activities should be as low as possible to obtain specific antimineralocorticoid compounds. In this paper, we describe two cellular models used for easy and specific measurement of mineralocorticoid and progestin activities. These models involve the induction of firely luciferase under hormonal control mediated by a chimeric receptor. The first model comprises transiently transfected MCF-7 cells, whereas the second uses stably transfected HeLa cells. Glucocorticoid activity was assayed with the classic tyrosine-aminotransferase induction method in HTC cells. Six compounds of a new family of 11β-substituted-17-spirolactone steroids were thus studied and compared to control compounds. Five of them showed antimineralocorticoid activity and one was active at a concentration lower than that of mespirenone.

Receptor binding affinity and antiproliferative activity of new antiinflammatory antedrugs: 6-methoxycarbonyl prednisolone and its derivatives

Systemic side effects of antiinflammatory steroids may be minimized by incorporation of a metabolically labile group which is metabolized to make the steroid inactive upon entry into the systemic circulation (antedrug concept). In continuing efforts to minimize systemic adverse effects of potent antiinflammatory steroids, we have recently synthesized methyl 11β, 17α,21-trihydroxy-3,20-dioxopregna-1,4-diene-6-carboxylate (P6CM), its 21-acetoxys (P6CMa, P6CMb) and 17,21-acetonide (P6CMacet) derivatives. Structure-activity relationships have now been assessed and compared with prednisolone (P) for glucocorticoid receptor affinity ( P IC 50 = 28 nM ), gluconeogenic activity as induction of tyrosine aminotransferase ( EC 50 = 4.4 nM) in H4-II-C3 HTC cells and antiproliferative effects ( P = 48% inhibition of [ 3H]thymidine incorporation at 1 μM). Relative potencies for receptor binding ( P = 1 ) were 0.12, 0.03, 0.004, and 0.0008 for P6CM, P6CMa, P6CMb, and P6CMacet, respectively, and enzyme induction relative potencies were 0.13, 0.05, 0.01, and 0.008, respectively. Antiproliferative effects of all derivatives were also less than that of P. These decreases suggest that addition of the 6-carboxymethyl group to prednisolone results in the general reduction of glucocorticoid activities. Taken together with previously reported results demonstrating retention of topical antiinflammatory activity of these novel steroids, P6CM and its derivatives may represent new locally active antiinflammatory steroids with reduced propensity to cause gluconeogenic and antiproliferative adverse effects.

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700–3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.