The effect of opioid peptides on corticofugal modulation of nociceptive response in thalamic ventrobasal nucleus (VB) produced by cortical sensorimotor area II (Sm II) was observed. Single unit activities of VB neurons were extracellularly recorded by glass microelectrodes. The results showed that the nociceptive responses of VB neurons were attenuated at 0-7.5 minutes after stimulating Sm II (n = 14, P < 0.05). The inhibitory effect of stimulation of Sm II was reduced after ventriculer microinjection of 20ul of naloxone (n = 9), while marked inhibition was shown at 0-2.5 minutes after stimulating Sm II in the saline control group (n = 9, P < 0.05). There was a statistical difference at 0-2.5 minutes after stimulating Sm II between the two groups (P < 0.05). These studies suggest that opioid peptides might be involved in corticofugal modulation of pain.

4-hydroxycarbophenyl retiamide (R II) is a new synthetic analogue of retinol, but with lower toxicity than retinoic acid. We studied its induction effects and its effects on some malignant phenotypes of the MFC cell line. The mechanism of these effects was also explored. MFC cells were grown in complete RPMI 1640 medium supplemented with 10(-5) mol/L R II for five passages. By then the cell growth rate slowed down; the rate of 3H-TdR incorporation and the colony-forming capacity of MFC cells decreased; morphologically, the cells became epithelial rather than fibroblastic with various degrees of polarization. Further investigation about the mechanism of these changes was also undergone. First by flow cytometry, it was shown that the R II-treated cells were retained in G1 phase. Second, dot blot hybridization showed a decrease of more than 61% of c-myc mRNA and an increase of more than 52% of v-fos mRNA. The major chromosome distribution changed from 54-56 to 46-54 with an increase in diploid. Scanning microscopic examination showed that the R II-treated cells were covered by numerous microvilli and pseudopodia with round terminal expansion in contrast to the ruffle protrusions and leaf-like pseudopodia of control cells. All the results suggested that R II could reverse some malignant phenotypes of MFC cells.

Pre-treatment of metastatic human lung cancer cell subline (PGCL3) with all trans retinoic acid (RA) resulted in inhibition of cell growth in vitro and invasion through the reconstituted basement membrane. RA was also noticed to inhibit the experimental metastatic ability of PGCL3. Data showed that 5/6, 3/6 and 2/6 of the nude mice developed lung colonization in the control, the 5 mumol/L and the 10 mumol/L RA treated PGCL3 cells respectively. Data from DNA-RNA dot blot hybridization further showed that the 10 mumol/L RA treated cells expressed high levels of the human tissue inhibitors of metalloproteinases (Timp-1 and Timp-2) in comparing to the untreated cells. These results may help to clarify the mechanism of RA-induced inhibition effect on tumor invasion and metastasis.

The promyelocytic leukemic cell line HL-60 with 20-200U/ml of low-dose rhTNF-a cultured in liquid culture system in vitro was used to observe the effect on HL-60 by TNF. TNF within dose of 50-200 U/ml can induce HL-60 cell differentiation along the monocytic-macrophage pathway, and inhibit HL-60 cell proliferation. The total RNA of HL-60 cell was used to hybrize to v-myc or v-fos probe by dot blot. We detected the expression changes of c-myc or c-fos proto-oncogene by 1-100U/ml of TNF inducing HL-60 cell for 1-12 hours. TNF could regulate the level of c-myc or c-fos mRNA, the transcription of c-myc was inhibited remarkdly, and the expression of c-fos was increased early. The results indicated that TNF in low-dose have effect on inducing HL-60 cell differentiation and regulating expression of multioncogene.

HL-60 cell lines and AML fresh bone marrow cells were incubated with rhTNF-alpha and rhIFN-gamma in suspension culture system. Then total RNA was prepared for dot blot with 32P nick-translated c-myc DNA probe. The expression changes of c-myc oncogene when the HL-60 cell lines were treated with rhTNF-alpha and IFN-gamma and the AML fresh bone marrow cells treated with rhTNF-alpha alone were observed. The results showed that when the HL-60 cells were treated with 100U/ml or 500U/ml IFN-gamma and 50U/ml rhTNF-alpha for 8 hours, the expression of c-myc oncogene can be inhibited remarkably. The combination of rhIFN-gamma and rhTNF-alpha shows synergistic effect on inhibition of c-myc expression. High expression of c-myc was found in 8 patients with AML; c-myc mRNA level decreased remarkably after treatment of fresh bone marrow cells with 50U/ml rhTNF-alpha for 12 hours in 6 cases, while the remaining 2 cases showed minimal changes. The results demonstrate that rhTNF-alpha have inhibitive effect on c-myc expression in HL-60 cells and AML fresh leukemic cells. It also indicates the possibility of treating AML with low-dose rhTNF-alpha.

This experiment was to study the mechanism of ATP anticancer effects. By using flow cytometry, Scrape-Loading and dye transfer (SLDT), dot hybridization methods, changes of cell cycle phase distribution, gap junctional intercellular communication (GJIC), and oncogene expression were observed in human stomach mucous glandular carcinoma (MGC-803) cells treated with ATP (0.23 mg/ml). It was found that ATP inhibited the proliferation and arrested cell cycle in S phase. The ATP-treated MGC-803 cells increased in GJIC, and decreased in expression of c-Ha-ras oncogene. These results indicated that the inhibition of proliferation and increased GJIC was closely correlated with the reduction of c-Ha-ras oncogene expression.

Using dot blot methods to identify the relationship between the oncogene expression and monosomy of chromosome No 15 in BHLB4 and their changes after induced differentiation, the results indicated that in Clone-1 (high frequency of chromosome No 15 monosomy) C-myc, H-ras, K-ras, N-ras and C-fos expression was higher than that in BHLB4. In Clone-4 (low frequency of chromosome No 15 monosomy), N-ras expression was lower than that in BHLB4. Decreased expression of C-myc, H-ras, and N-ras oncogenes could also be observed during cell differentiation induced by buta acid. It is possible that an antioncogene located on chromosome No 15 regulates oncogene expression, and differentiation agents would have obvious effects on it.

Effect of selenium on the protein kinase c-alpha (PKC-alpha) gene expression was observed in the preneoplastic lesions in liver of S.D. rat Solt-Farber model. Overexpression of PKC-alpha was found in preneoplastic liver but not in partially hepatectomized liver. Administration of 4PPm Na2SeO3 in water 2 weeks before i.p injection of DEN (200 ng/kg bw) for 8 weeks reduced the PKC-alpha gene overexpression in preneoplastic liver and the formation of hyperplastic foci, alpha-GT-altered foci and preneoplastic lesions.

In order to study the inhibitory effect of antisense oligodeoxynucleotides on the growth of human pancreatic adenocarcinoma cell lines (PC-2 & PC-3), synthesized c-myc & c-Ki-ras antisense oligodeoxynucleotides (ASODN) and sense oligodeoxynucleotides (SODN) were added to the culture media of PC-2 & PC-3 cell lines. The cell growth activities after 24, 48, 72 hours of culture were estimated by cell count and 3H-TdR incorporation, and the concurrent oncogene expressions were studied by adopting reverse transcription PCR technique. Cell growth was significantly inhibited after exposure to ASODN for 24 & 48 hours. Cell growth in the c-Ki-ras ASODN group approached nearly the same level as in the control groups 72 hours after exposure, whereas cell proliferation in the c-myc ASODN medium was still being inhibited. By reverse transcription PCR technique, marked inhibition of c-myc & c-Ki-ras expression was seen in the groups after 24 & 48 hours treatment of c-myc & c-Ki-ras ASODNs. There was also down-regulation of c-myc & c-Ki-ras expression after 72 hours exposure to both oncogene ASODNs.

The expression of p53 gene has been found to be regulated during the induction of differentiation of U937 leukemic cells into mature macrophages by recombinant human granulocyte- macrophage colony stimulating factors (rhGM-CSF) We showed here that the increased expression of p53 seemed to be necessary for the differentiation of U937 cells induced by rh-GM-CSF. The inhibition of p53 expression by a p53 antisense oligodeoxynucleotide lead to the significant decrease of formation of mature macrophages from U 937 cells in the presence of rhGM-CSF. By contrast, the p53 sense oligodeoxynucleotide had no any effect. Furthermore, we have analysed the growth of U937 cells in the presence or absence of rhGM-CSF. The results showed that rhGM-CSF dramatically inhibited the growth of U 937 cells in the cultures. At the same time, the antisense inhibition experiment demonstrated that the inhibition of p53 expression partially diminished the growth-inhibitory effect of rhGM-CSF on U 937 cells. These results suggested that the p53 was required for the initiation of rhGM-CSF-induced differentiation of U 937 cells on one hand, and the inhibition of cell growth on the other hand. Thus we deduce that the increased expression of p53 induced by rhGM-CSF may be a coupling event of switch of U 937 cells from growth into differentiation.

Dimethylbenzanthracene (DMBA) stimulates expression of P1-450 gene in human lung tumor cells (ChaGo). A concentration and time-dependent increase in the level of P1-450 specific mRNA sequences has been observed in ChaGo cells treated with sublethal concentrations of DMBA. The methylation pattern of "-CCGG-" sequence of most of the coding region and of the 3' end of P1-450 gene is not affected by such DMBA treatment; DMBA-treatment of the cell induces hypomethylation of only the 5' end "-CCGG-" sequences of the gene. These results demonstrate that DMBA-treatment of ChaGo cells induced increased expression of gene can be correlated to the increased degree of site specific hypomethylation of the internal "-C-" residues of the "-CCGG-" sequences located at the 5' end of the P1-450 gene. However, it is also possible to have other molecular mechanism of regulation.

To clarify the effects of female sex hormones on ANF gene expression, emasculated and intact female rats were subcutaneously injected with estradiol (E2), progesterone (P) and olive oil solvent (O) respectively, once a day for 7 days. The relative rANF-mRNA contents of rat atria were measured by molecular hybridization, and rANF-cDNA was labeled with alpha-32P as probe. The groups and hormone doses per 100 g body weight were: (1) sham, 0.2 ml O; (2) rats were bilaterally ovariectomized(OVX), 0.2 ml O; (3) OVX, 80 ng E2; (4) OVX, 80 micrograms E2; (5) OVX, 0.2 mg P; (6) OVX, 2 mg P; (7) OVX, 80 ng E2, 0.2 mg P. The results showed that the rANF-mRNA of group (2) was the lowest, it was 75% of that in group (1), while the rANF-mRNA levels in groups (3)-(7) were 1.1, 2.0, 1.9, 2.3 and 2.1 folds of that in group (2) respectively. The results revealed that E2 and P increased ANF gene expression. Their effects were associated with the dosage. The two may have synergistic actions. Physiological amount of E2 and P may maintain suitable level of rANF-mRNA. This experiment suggested that female sex hormone may have dual effects in body fluid balance.

Experiments were conducted in intact and adrenalectomized female Wistar rats given dexamethasone, deoxycorticosterone or both, and atria total RNA was extracted with cold phenol method and hybridized to rat ANP-cDNA probe labeled with alpha-32P. The ANP gene transcripts in intact rats receiving dexamethasone and in adrenalectomized rats receiving dexamethasone and deoxycorticosterone increased by 2-fold, while there were no significant changes in the other groups. The results suggest that glucocorticoids enhance ANP gene expression, an effect relying on the presence of mineralocorticoids which given alone does not give rise to ANP gene expression enhancement.

The specific binding sites for atrial natriuretic peptide (ANP) were present in cultured vascular smooth muscle cells (VSMC) from stroke-prone spontaneously hypertensive (SHRsp) and Wistar-Kyoto(WKY) rats with a Bmax of 3.65 +/- 0.13 and 1.89 +/- 0.09 pmol/mg pr. and a Kd of 72.0 +/- 10.2 and 42.1 +/- 4.8 x 10(-12) mol/L, respectively. The basal levels of cGMP of the two strains showed no statistical difference. After treatment with ANP (1.67 x 10(-7) mol/L) for 5 min, the cGMP levels of VSMC were increased by 139 folds in SHRsp and 271 folds in WKY rats, i.e., cGMP levels were significantly lower in the former (P less than 0.01). Therefore, the cultured VSMC of SHRsp had higher ANP receptor density but lower affinity and responsiveness to ANP than that of WKY rats. After incubation of VSMC in the medium containing high NaCl (2-folds of normal) at 37 degrees C for 24 h, the number of ANP binding sites decreased to 34.8 +/- 8.2% in SHRsp and to 38.6 +/- 9.4% in WKY rats (P less than 0.01) with a parallel decrease of cGMP, while the affinity of ANP receptor did not change. It is suggested that the lower responsiveness of ANP receptor to ANP in SHRsp might result in a diminution of vasorelaxation to ANP and thus an increase of arterial pressure. In addition, the more down-regulation of ANP receptor by high NaCl in cultured VSMC from SHRsp implicates that it is one of the mechanisms that high dietary intake of NaCl might enhance high blood pressure.

Modulation of a number of protooncogene expression occurs during differentiation of eukaryotes. Changes in expression of c-myb and c-myc during the HMBA-induced terminal differentiation of murine erythroleukemia cells were characterized by an early decrease (within 4 hrs), followed by the recovery of c-myc mRNA by 10 hrs, and the retention of suppression of c-myb expression for the rest of the induction period. Two MELC variants were used to further define the relationship between the differentiation and the protooncogene expression. R1 was a MELC variant completely resistant to HMBA, and R1 (VCR), a vincristine resistant R1, became inducible by HMBA again. The cell differentiation and the c-myb and c-myc expression were determined on R1 or R1 (VCR) cultured with HMBA respectively. The results demonstrated that the c-myc mRNA increased and remained relatively high as the cells grew to a saturated density regardless of the induction of differentiation. The R1 (VCR) cultured with HMBA displayed an early decrease in c-myb mRNA and a subsequent suppression of its expression, while the R1 cultured with HMBA showed a stable level of c-myb mRNA. These results suggest that the c-myb expression, rather than c-myc expression, is closely related to the HMBA-induced terminal differentiation.

In this paper, the immunoregulatory effects of calcium ionophore A23187 on the accessory function of mouse spleen dendritic cells (DC) in primary mixed lymphocyte reaction (1.MLR) and oxidative mitogenesis are reported. A23187 had a direct enhancing effect on DC. When pretreated with 0.5-1 mumol/L A23187 for 6-8 h, the accessory activity of DC was twice as high as that of the control. This is circumstantial evidence indicating an up-regulation of activity mediated by an increase in intracellular Ca2+ levels and PKC activation.

The effect of azelastine, a new on the down-regulation of beta-receptor agonist was investigated. Male Hartley guinea pigs received injections of saline or terbutaline (T.) and/or azelastine (A.) for successive 7 days. The radioligand binding assays for beta-adrenoceptors in the lung membranes of the guinea pigs were performed. The results showed the differences of numbers of maximal binding sites (Bmax) among four groups were significant. The Bmax of beta-adrenoceptor in T. group was less than that in control group (P less than 0.02). The Bmax in A. group was more than that in control group (P less than 0.05). The Bmax in T. plus A. was more than that in T. group, and there was no significant difference between Bmax in T. group and in T. plus A. group (P greater than 0.1). The differences of affinity (Kd) of beta-adrenoceptor among four groups were not significant. Azelastine increased the density of beta-adrenoceptors and partially prevented the down-regulation of beta-adrenoceptor caused by terbutaline.

Our studies show that the growth of transplantable chondrosarcoma in rats is inhibited significantly by N-4-(hydroxycarbophenyl) retinamide (RII) at a dosage of 50 mg/kg. RII show a weak ability to induce the differentiation of HL-60 cells at a concentration of 1 umol/L. However, most of the HL-60 cells were induced to differentiate along granulocyte lineage after exposure to RII (1 mumol/L) and S86019 (10 micrograms/ml). Flow cytometry studies indicated that the majority of HL-60 cells were arrested in G1 phase by RII plus S86019. Northern blot analysis clearly demonstrated that c-myc expression was inhibited after treating HL-60 cells with RII plus S86019 for 12 hours. Moreover, thymidylate synthetase mRNA transcription was inhibited in those differentiated cells.