The expression of HLA class II molecules is mainly regulated transcriptionally and this regulation is thought to play an important role to the control of immune response. In this report, we have studied the effect of adenylate cyclase activator, forskolin, to the expression of HLA class II molecules on the cell surface of an human multiple myeloma cell line, RPMI8226. On the northern blot analysis and FACS analysis, we have revealed that forskolin upregulated the expression of mRNAs of DQB and DRB gene and their products on its surface. On the sequence analysis of upstream of HLA-DQB gene, we have identified not only Y-,X-, W-box, which were thought to regulatory region of truncated gene, but also cAMP responsible element (CRE) like regulatory region, which located upstream of W-box. On the gel retardation assay, when we used DNA probes that were specific for CRE like region and Y-box, we have found newly detectable bands, which appeared by forskolin treatment. These data suggest that forskolin upregulates HLA class II molecules by means of the interaction between CRE and cAMP responsible element binding protein (CREB).

Methylprednisolone (MP), a prototype of glucocorticoids, was administered to patients with shock, low output syndrome and cardiac failure who had required long-term infusion of dopamine (DOA) and/or dobutamine (DOB), when they showed signs of desensitization to catecholamines (CA). Subjects were 141 patients selected from 37 hospitals. The mean infusion periods were 7.0 days with DOA and 7.5 days with DOB. MP elevated systolic and diastolic blood pressure significantly for 1 to 6 hours, concomitantly with increased urinary output. The results suggest that MP could attenuate down regulation of adrenoreceptors induced by long-term CA infusion.

Prostaglandin (PG) endoperoxide synthase is a bifunctional enzyme with fatty acid cyclooxygenase activity (arachidonic acid----PGG2) and PG hydroperoxidase activity (PGG2----PGH2). The primary structure of the enzyme was determined recently by cloning and sequencing the cDNAs for sheep and mouse enzymes and the genomic DNA for the human enzyme. Aspirin selectively inhibits the fatty acid cyclooxygenase activity but not the PG hydroperoxidase activity by acetylating the serine #506. Several lines of evidence suggest that cyclooxygenase enzyme is inducible by several biofactors, such as EGF, TGF-beta, IL-1, and epinephrine. Furthermore, it has been recently suggested that glucocorticoids inhibit the synthesis of the enzyme by the conversion of cyclooxygenase mRNA into a cryptic, untranslatable form.

We have examined the effect of verapamil on CsA-induced nephropathy by measurement of systemic hemodynamics including each organ blood flow using the microsphere method in ischemic kidney model of hemi-nephrectomized Wistar rats. Hepatic and renal microsomal cytochrome P-450 contents and their enzyme activities were measured to study the correlation between CsA-induced nephropathy and induction of hepatic and renal microsomal cytochrome P-450. All rats were hemi-nephrectomized (l-nephrectomy) and were classified into the following 6 groups: 1) control groups, 2) CsA at a dose of 40 mg/kg per day orally for 7 days (CsA group), 3) Oral administration of verapamil for 7 days in the CsA group (CsA + V group), 4) 20 min clamping of the remaining right kidney pedicle (Ischemic, Is group), 5) CsA was administered in the Is group (Is + CsA group), 6) Addition of verapamil to CsA in the Is + CsA group (Is + CsA + V group). Verapamil was given in the drinking water and the average dose calculated from the amount of drinking was 4.7 +/- 1.0 mg/kg per day and 5.2 +/- 0.7 mg/kg per day for the CsA + V group and the Is + CsA + V group, respectively. CsA caused significant increases in BUN and serum creatinine (sCr) with a significant decreases in renal inulin clearance (CIn) in all groups. When compared with the Is group, CsA caused significant decreases in cardiac output and all organ blood flow especially in renal blood flow with significant increases in BUN and sCr in the Is + CsA group.2+ degree of nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute toxicity, inductive effects of liver enzymes and liver persistency of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PenCDD) were compared with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using male Wistar rats. 1,2,3,7,8-PenCDD treatment at a dose of 0.1 mumol/kg resulted in significant depression of growth of rats from a day to 28 days after treatment. However, the effect was relatively less than that of 2,3,7,8-TCDD. On 5 days, similarly to 2,3,7,8-TCDD-treated group, liver hypertrophy and thymic atrophy were observed in 1,2,3,7,8-PenCDD-treated groups. In addition, 1,2,3,7,8-PenCDD showed potent 3-methylcholanthrene-type inducing ability. For example, the activities of benzo(a)pyrene 3-hydroxylase and DT-diaphorase were 25-fold and 10-fold of control, respectively. On 30 days, about 50% of the inductive effects on 5 days were maintained in both 1,2,3,7,8-PenCDD- and 2,3,7,8-TCDD-treated groups. Amount of 1,2,3,7,8-PenCDD distributed to the liver on 5 days was about 80-90% of dose and was about 1.5 times greater than that of 2,3,7,8-TCDD. About 50% of dose of 1,2,3,7,8-PenCDD remained even on 30 days after treatment. From these results, it is suggested that 1,2,3,7,8-PenCDD possessing the potent acute toxicity comparable to 2,3,7,8-TCDD and higher persistency in the liver might be more important than 2,3,7,8-TCDD in terms of the chronic toxicity.

Immunohistochemical determination of 3-methylcholanthrene (MC) inducible cytochrome P-450 (MC-P-450) was investigated in rat and human tissue, and its clinical availability was discussed. Induced by MC, MC-P-450 in rat liver and stomach was well stained immunohistochemically, showing clear contrast against control without induction. The staining intensity in the tissue was correlated with the amount of tissue MC-P-450 which was determined previously by electrophoretical and biochemical technics. By the same immunohistochemical method MC-P-450 in human liver and stomach was also detectable. The staining grade of MC-P-450 in human liver and stomach was different from each person. However, its intensities in liver and stomach in the same individual showed clear correlation with p less than 0.02. Since MC-P-450 in liver plays a major role in drug metabolism, the proof of correlation between staining degree of the resected stomach and hepatic tissue would provide useful clue in gastric cancer for postoperative administration of masked compounds activated by MC-P-450.

The regulation of collagenase gene expression in the human osteosarcoma-derived osteoblastic cell lines MG-63, U2-OS and human fibroblast cell line IMR-90 was investigated by Northern blot analysis. Exposure of quiescent MG-63, U2-OS and IMR-90 cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 10% fetal calf serum (FCS) resulted in the induction of mRNA encoding collagenase. Epidermal growth factor (EGF) induced collagenase mRNA in the IMR-90 cell but not in the MG-63 and U2-OS cells. In the IMR-90 and MG-63 cells, EGF stimulated the transcription of the c-fos and c-jun genes encoding the transcriptional factors which interact directly with the promoter region of the human collagenase gene. Parathyroid hormone and 1,25-dihydroxy-vitamin D3 did not increase the collagenase mRNA level in both osteosarcoma cells. Recombinant interleukin-1 beta (rIL-1 beta) induced collagenase and c-jun but not c-fos mRNA in the MG-63 cell. The induction by rIL-1 beta was blocked by cycloheximide and dexamethasone. Transforming growth factor beta 1 blocked the FCS-induced collagenase gene expression but partially inhibited the rIL-1 beta-induced gene expression in the MG-63 cell. These results suggest that the collagenase activity is regulated not only by post-translational modification but also at the transcriptional level by the various factors in bone.

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.

The hamsters have been known to be the least sensitive mammalian species to the acute toxicity of highly toxic polyhalogenated hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the present study, the tissue distribution, inductive effect of liver enzymes and acute toxicity of 2,3,4,7,8-pentachlorodibenzofuran (PenCDF) in male Golden Syrian hamsters were examined. The highest content (about 48% of dose) of PenCDF was found in the liver 5 days after a single i.p. dose of 1.0 mg/kg. The amount ranging about 5 to 10% of dose was also distributed to mesentery, skin and muscle. In liver, the distribution of PenCDF was just parallel to that of cytochrome P-450 (P-450), marker enzymes of liver endoplasmic reticulum, suggesting that PenCDF binds to P-450. The mode of inductive effects of PenCDF in hamsters was 3-methylcholanthrene-type as reported previously in rats. However, the typical enzymes such as benzo(a)pyrene 3-hydroxylase and DT-diaphorase were induced to a relatively less extent than did in rats. In hamsters pretreated with PenCDF at a dose of 0.5 mg/kg, the potent atrophy of thymus and the 3-fold increase of liver lipid peroxide were observed, whereas the body weight gain was not suppressed at all. These results suggest that the induction of liver enzymes and the atrophy of thymus might not be the direct cause of PenCDF-induced lethality in hamsters.

PCBs, non-ortho chlorine substituted PCBs (Co-PCBs), PCQs and (PCDFs + PCDDs), all of which contained similar compositions of those corresponding in yusho oil, were prepared from a PCB preparation used as a heat exchanger fluid. After dissolved in 1, 4-dioxane, they were applied into the air sac of white leghorn eggs incubated for 16.5 days at 37.5 degrees C. Forty eight hours after injection, the hepatic benzo(a)pyrene hydroxylase (AHH) and 7-ethoxyresorufin deethylase (EROD) activities were assayed. The average relative potencies of induction for the two microsomal drug metabolizing enzymes by (PCDFs + PCDDs), Co-PCBs, PCBs and PCQs were 100, 13.4, 0.0006 and 0.0004, respectively. The toxic effects for yusho disease by these substances were calculated from the relative enzyme induction potencies and the average concentrations in yusho oils with the production dates of February 9 and 10, 1968. Consequently, the relative toxicities of (PCDFs+PCDDs), Co-PCBs, PCBs and PCQs were 100, 13.2, 0.06 and 0.12, respectively. This result, as well as our previous investigations using rats and monkeys, insists that (PCDFs+PCDDs) are the primary causal agents for yusho disease. However, the Co-PCBs, which were recently detected in the yusho oils by us, were revealed to be fairly effective in yusho manifestation. In addition, it was cleared that the hepatic enzyme induction by the Co-PCBs fraction, which contained other several PCB isomers, was almost completely contributed by only Co-PCBs such as 3,4,3',4'-tetra- 3,4,5,3',4'-penta- and 3,4,5,3',4',5'-hexachlorinated biphenyls present in the fraction. A chemical uptake rate from the air sac by the chick embryo decreased significantly in the cases of extremely high doses of PCBs (10,000 micrograms/egg) and PCQs (3,333 and 10,000 micrograms/egg), and result the elevations of hepatic enzymes activities were depressed, indicating that the suitable chemical dose amount to be less than about 1,000 micrograms/egg.

Human papillomaviruses (HPVs) are known as etiologic agents of various diseases in regions of the human epithelium. Specific type HPV16 is most frequently found in association with human squamous cell carcinoma. To examine biological activity of HPV type 16 in human cells, primary foreskin epidermal cells and dermal fibroblasts were transfected by recombinant viral DNA containing neo gene with Ca2+-phosphate precipitation. Epidermal cells were maintained in 0.5% Chelex-treated fetal calf serum, low calcium medium supplemented with bovine pituitary extracts and hormone mix. Fibroblasts were cultured in DMEM plus 10% fetal calf serum. The transfected cells were then selected with G418-resistant phenotype. These cells were propagated to maintain in culture and subsequently became stable lines carrying HPV16 genomes, while mock transfected control cells died off at approximately 40-50 population doublings (PD) in a parallel experiment. We have established two independently immortalized human epidermal cell lines (PHK16-I and II) which harbor different copies of HPV16 genome and express HPV16 specific mRNA. Although younger populations of PHK16 lines were fairly sensitive to high Ca2+-level to be differentiating keratinocytes, progressive changes of the cellular phenotype were demonstrated in terms of changes in Ca2+-response and anchorage independent growth during over 300 PD. Altered Ca2+-regulation of growth and differentiation appeared to be common reliable phenotype associated with stable transformation of skin epidermal cells. In contrast, none of the HPV16-transfected fibroblast line immortalized but simply showed extended life span up to 100 PD in average. The result suggested that this biological activity of HPV16 could be reflected in HPV-tropism related to epithelial transformation. We then studied correlations between HPV16 gene expression and the regulation of growth and differentiation of PHK lines during the progressive transformation. Northern blot analysis of RNA from cells in earlier passages demonstrated that down-regulation of HPV16 E6/E7 transcription was associated with keratinocyte differentiation induced by added 1.0mM calcium. The p97 promoter for HPV 16 early genes covering E6/E7 was specifically responsible for this Ca2+-regulation. The eventual loss of Ca2+-regulation could be implicated in a process of progressive transformation of HPV16-epidermal cell system.