Inhibition Of Thymidine Incorporation Research Articles

Biodegradable microspheres containing a colchicine analogue inhibit DNA synthesis in vascular smooth muscle cells.

Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Local application of agents capable of modulating vascular responses, including smooth muscle cell proliferation, has been achieved, but difficulty in maintaining active levels locally has been a factor limiting the efficacy of such approaches. One strategy to maintain adequate levels is the local delivery of microspheres that release active agents over sustained time periods. We incorporated a colchicine analogue into biodegradable microspheres composed of a lactic acid/glycolic acid copolymer and characterized their drug release behavior as well as their effects on bovine aortic smooth muscle cells (BASMCs) in culture. Drug release was evaluated by spectrophotometric assay. Drug effects on DNA synthesis were measured by thymidine incorporation after addition of serum to subconfluent cells synchronized by serum withdrawal as well as in asynchronous cell populations. Polymeric microspheres incorporating 10% to 17% drug by weight and averaging 6 microns in size were found to release the colchicine analog in buffered saline solutions over more than several weeks. Drug-loaded particles inhibited DNA synthesis completely, with EC50 values ranging from 0.001 to 0.005 g% (wt/wt). Morphological changes suggesting microtubule depolymerization were observed after drug particle treatment, with similar EC50 values. Microspheres allowed to contact the cell surface demonstrated effects similar to those seen with microspheres suspended in the nutrient medium by porous polycarbonate filters, at EC50 values approximately fivefold lower. In contrast, control microspheres composed only of polymer with no incorporated active drug demonstrated no observable toxicity to BASMCs and < 40% inhibition of thymidine incorporation even in suspensions containing up to 0.5 g% particles. Biodegradable microspheres were fashioned that release a colchicine analogue and inhibit DNA synthesis in smooth muscle cells. Drug-loaded polymeric particles are candidates for local delivery at sites of arterial injury to decrease restenosis.

PMA Inhibits the Growth of Human Fibroblasts after the Induction of Immediate-Early Genes

WS-1 cells, human fibroblasts derived from embryonic skin, become quiescent in serum-free medium. Addition of serum or thrombin stimulates incorporation of 3 H-labeled thymidine 16-40 h later. The tumor promoter phorbol 12-myristate 13-acetate (PMA) inhibited this response in quiescent cells stimulated with serum or thrombin. PMA also inhibited thymidine incorporation in rapidly growing WS-1 cells. Since the inhibition caused by PMA could result from activation of protein kinase C (PKC) or the down-regulation of PKC, the effect of the PKC inhibitor, GF 109203X, was tested. GF 109203X did not affect thymidine incorporation induced by serum. GF 109203X prevented the inhibition caused by PMA, suggesting that the activation of PKC is required for the inhibition of DNA synthesis. A prolonged activation of PKC is probably required, since a single treatment of a synthetic diacylglycerol does not inhibit and addition of GF 109203X 2 to 8 h after PMA reverses the inhibition. One possible target for PMA activity is the induction of immediate-early response genes. Thrombin or serum treatment of quiescent WS-1 cells induced protooncogenes c- fos and c- jun. PMA also induced c- fos and c- jun with a time course similar to that of serum or thrombin. The induction of c- fos by PMA was sensitive to staurosporine or GF 109203X. A time course of PMA addition indicated that maximal inhibition of DNA synthesis occurred when PMA was added 4 h after stimulation with serum. These results suggest that the PMA-induced inhibition of DNA synthesis occurs through an activation of PKC that inhibits DNA synthesis at a point after the induction of c- fos and c- jun.

Sex difference in the action of activin-A on cell proliferation of differentiating rat gonad.

The effect of recombinant bovine activin-A on DNA synthesis in fetal rat gonads and mesonephroi was studied in vitro by using [3H]thymidine autoradiography to evaluate the role of activin in the regulation of gonadal development. mRNA levels of inhibin-beta A and -beta B, activin receptor II and IIB (ActRII and ActRIIB), and follistatin were studied by Northern hybridization in the fetal rat gonads and mesonephroi. Activin stimulated thymidine incorporation in ovaries and female mesonephroi on days 14 and 15 postcoitum (pc) in a dose-dependent manner, as assessed by autoradiography. In contrast, activin inhibited thymidine incorporation in testes and male mesonephroi on day 14 pc in a dose-dependent way. On day 18 pc, the stimulatory effect of activin in ovary was nearly lost, whereas that in mesonephros was still pronounced. Activin had no effect on thymidine incorporation in testes and male mesonephroi on day 15 or 18 pc. Inhibin-beta A mRNA was seen in testes and mesonephroi in the male and in mesonephroi in the female of 15 and 18 day pc embryos. Inhibin-beta A mRNA expression was also seen in the testes and epididymes of newborn male rats and in the ovaries of 11-day-old postnatal rats. Inhibin-beta B mRNA was present in both testes and ovaries from 15 day pc onward. ActRIIB mRNA was most abundant in the testes, ovaries, and mesonephroi of 15 day pc embryos. The expression of ActRIIB message diminished during aging. ActRII mRNA was ubiquitously expressed during development. Follistatin mRNA was seen in the ovaries from 15 day pc onward and in the oviducts at 11 days postnatally. The present novel findings of activin subunit and receptor mRNA expression and activin action on gonadal and mesonephric cell proliferation suggest an important, sexually dimorphic role for this substance in fetal gonadal differentiation.

Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B.

Natriuretic peptide receptor-B (NPR-B) was identified in rat chondrocytes, and its physiological functions were investigated. Rat tissues, including the xiphoid cartilage, brain, lung, liver, adrenal gland, and kidney, were screened for NPR-B activity, which we assayed by receptor guanylate cyclase activity specifically stimulated by C-type natriuretic peptide (CNP), a known selective activator of NPR-B. Cartilage showed distinctly higher NPR-B activity. Furthermore, exposure of cultured rat chondrocytes to CNP (10(-6) M) resulted in a large increase in intracellular cGMP production (376 +/- 38 pmol/well), with threshold responses occurring between 10(-10) and 10(-9) M CNP. Atrial natriuretic peptide and brain natriuretic peptide also stimulated cGMP production in rat chondrocytes but with a potency that was at least 10 times less than that of CNP. Polymerase chain reaction analysis also demonstrated NPR-B gene expression in adult rat xiphisternum and cultured chondrocytes. These findings indicate that NPR-B is present in rat chondrocytes. In rat chondrocytes exposed to CNP, [3H]thymidine incorporation was inhibited in a dose-dependent manner (half-maximal response, 10(-11)M). However, much higher concentrations of atrial natriuretic peptide were required to induce the inhibition of thymidine incorporation. Interestingly, CNP-like immunoreactivity was detected in the conditioned medium from chondrocyte cultures. In addition, TGF-beta 1, a multifunctional cytokine, induced a marked increase in CNP secretion and CNP mRNA levels in chondrocytes. These results indicate that autocrine CNP inhibits mitogenesis in chondrocytes via NPR-B under the control of TGF-beta 1.

Toxicity of ifosfamide, cyclophosphamide and their metabolites in renal tubular cells in culture.

Ifosfamide (IF) and cyclophosphamide (CP) are highly effective alkylating cytostatic drugs. IF and CP have to be activated through a metabolic step in vivo; numerous metabolites are known. While both IF and its structural isomer CP have severe urotoxic side effects, only IF is also a nephrotoxic drug, causing tubular damage resulting in Fanconi syndrome in some cases. Little information is available regarding the pathogenic mechanism of tubular damage by IF. We used the renal epithelial cell line LLC-PK1, which has many properties of the proximal tubule, in order to investigate the toxicity of IF and CP and of their reactive metabolites 4-hydroxy-IF (4-OH-IF), 4-hydroxy-CP (4-OH-CP), acrolein and chloroacetaldehyde (CAA). Protein content of monolayers, DNA and RNA synthesis were determined by standard techniques (thymidine and uridine incorporation). IF and CP had the lowest toxicities of all compounds tested. Both drugs inhibited thymidine incorporation by about 30% at a concentration of 300 mumol/l after 1 h incubation. 4-OH-IF and 4-OH-CP were significantly more toxic than the parent drugs. Thymidine incorporation, the most sensitive parameter, was reduced by about 70% by 300 mumol/l of either compound. In addition, 4-OH-CP reduced the total protein content of monolayers. 4-OH-IF did not effect protein content and RNA synthesis. Acrolein, the most toxic metabolite tested, reduced all three parameters significantly at concentrations of 50-75 mumol/l after 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

On the formation of homo‐azasteroidal esters of N,N‐bis(2‐chloroethyl)aminobenzoic acid isomers and their antitumor activity

AbstractThe N, N‐bis(2‐chloroethyl)aminobenzoate isomers and the 4‐methyl‐3‐N, N‐bis(2‐chloro‐ethyl)aminobenzoate of 3β‐hydroxy‐13α‐amino‐13,17‐seco‐5α‐androstan‐17‐oic‐13,17‐lactam, 3α‐hydroxy‐13α‐amino‐13,17‐seco‐5α‐androstan‐17‐oic‐13,17‐lactam, 3α‐hydroxy‐13α‐amino‐13,17‐seco‐5‐androsten‐17‐oic‐13,17‐lactam and 17β‐hydroxy‐3‐aza‐A‐homo‐4α‐androsten‐4‐one, have been prepared and their biological activity evaluated against P388 leukemia in vivo and Ehrlich Ascites tumor (EAT), P388 and L1210 leukemias and Baby Hamster cells (BHK) in vitro. The esters in which the alkylating congener is linked to the lactam alcohol in the axial position are inactive in vivo in P388 leukemia, while compounds 1, 4, 6, 13, 14 and the alkylating congeners 17, 18 and 20 are active. The effect of the homo‐azasteroidal of N, N‐bis(2‐chloroethyl)aminobenzoic acid isomers and of 4‐methyl‐3‐N, N‐bis(2‐chloroethyl)aminobenzoic acid on the incorporation of the radioactive precursor into the DNA of L1210, P388 leukemias, Ehrlich ascites tumor and, baby Hamster kidney cells was investigated. Higher inhibitory effects on the incorporation of the radioactive precursor was obtained with the ortho derivatives, yielding &lt;70% inhibition of thymidine incorporation in all tumor lines tested.

Induction of ornithine decarboxylase activity in mouse B lymphocytes.

We have examined the role in B cell activation of ornithine decarboxylase (ODC), the labile rate-limiting enzyme in the synthesis of polyamines thought to be required for S phase entry in all cells. When small resting mouse splenic B cells were stimulated with the mitogenic agents phorbol myristate acetate (PMA) plus ionomycin (lo), LPS or the B cell specific agent F(ab')2 anti-lg, ODC activity was greatly increased. ODC activity in small dense B cells remained near baseline levels for the first 6 h after treatment with LPS, but then increased approximately 150-fold in the next 18 h. When purified B cells were not separated by cell density, ODC activity was 30-fold greater at baseline and rose earlier after LPS stimulation, reaching a level about three times that of LPS-stimulated small, dense B cells at 24 h, implying that large (preactivated) B cells have much greater ODC responses than small, dense B cells. ODC activity, like S phase entry, could also be induced in small, dense B cells by PMA and lo but failed to respond to either agent alone. ODC levels rose transiently by approximately 40-fold between 2 and 6 h following stimulation of small B cells with F(ab')2 anti-lg, then declined to baseline. Whole anti-lg did not stimulate ODC activity and also blocked the F(ab')2 anti-lg mediated increase in ODC activity, just as it produced the expected inhibition of thymidine incorporation and cellular progression into S phase.(ABSTRACT TRUNCATED AT 250 WORDS)

TM-1 cells from an established human malignant glioma cell line produce PDGF, TGF-alpha, and TGF-beta which cooperatively play a stimulatory role for an autocrine growth promotion.

We have previously established a human malignant glioma cell line, TM-1. TM-1 cells could proliferate in the serum-free medium. In the present study, immunochemical analysis demonstrated that platelet-derived growth factor (PDGF), transforming growth factor (TGF)-alpha, and TGF-beta are present in the serum-free medium conditioned by growing TM-1 cells. While the cells appeared to possess a single type of binding sites for epidermal growth factor (EGF) with properties comparable to those determined for other tumor cells, the conditioned medium did not contain EGF.PDGF, TGF-alpha, and EGF added exogenously to serum-free media stimulated thymidine incorporation into DNA of TM-1 cells. In addition, antibodies specific for PDGF and TGF-alpha suppressed this activity. These results indicate autocrine and stimulatory roles of PDGF and TGF-alpha for the proliferation of TM-1 cells. As observed for other tumor cells, TGF-beta by itself weakly suppressed thymidine incorporation by TM-1 cells. However, TGF-beta employed in combination with TGF-alpha or EGF appeared to stimulate thymidine incorporation, suggesting that a cooperative action of TGF-beta with different growth factors may be involved in the stimulatory growth regulation at least for TM-1 cells.

HL-60 cell differentiation induced by phorbol- and 12-deoxyphorbol-esters.

The human promyelocytic leukaemia cell (HL-60) undergoes differentiation into a macrophage-like form when exposed to both tumour promoting- and non-promoting phorbol esters. We have investigated the effect of the two non-promoting phorbol esters, 12-deoxyphorbol-13-O-phenylacetate (Dopp) and 12-deoxyphorbol-13-O-phenylacetate-20-acetate (Doppa) on HL-60 cultures, and compared them with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All phorbol esters tested were found to be able to stop HL-60 proliferation and induce cell adherence and morphological changes characteristic of differentiation. TPA, fully differentiating at 1 nM, was more potent than Dopp and Doppa, which required 100 nM for full differentiation effects within the 4 day study. Doppa initially appeared weaker than Dopp at inhibiting incorporation of thymidine, the earliest effect studied, but we were able to detect rapid C-20 deacylation of Doppa, converting it to Dopp, using an HPLC protocol presented here. A detailed study of this thymidine incorporation inhibition showed that both TPA (10 nM or greater) and Dopp (500 nM or greater) have very similar time courses, with 50% inhibition occurring at approximately 12 h, in contrast to Doppa which had a significantly delayed time course at all doses tested. Exposure tests indicated that Dopp and Doppa could be washed from the cells much more easily than TPA. The data presented here strongly support the notion that the metabolic conversion of Doppa to Dopp by HL-60 cells was necessary to mediate its differentiating effects. Since protein kinase C (PKC)-beta 1, present in HL-60 cells, has been found to be the only PKC isotype activated so far in vitro by Doppa, our results suggest that activation of this isotype is not sufficient to drive HL-60 differentiation in vivo.

Neither cyclosporin A nor FK506 affects adenylate cyclase responses of fetal rat keratinizing epidermal cells (FRSK cells) at concentrations that inhibit thymidine incorporation

It has been reported that various anti-psoriatic agents augment the beta-adrenergic adenylate cyclase response that is defective in the psoriatic hyperproliferative epidermis. Recent evidence indicates that cyclosporin A (CsA) and FK506 show beneficial effects on psoriasis. Using fetal rat keratinizing epidermal cells (FRSK cells) we investigated the effects of these compounds on the adenylate cyclase system. These immunosuppressants had no effect on the adenylate cyclase responses of FRSK cells up to 1–10 μM concentration, although they significantly inhibited thymidine incorporation at concentrations more than 0.1 μM. There was no significant difference in the inhibitory effect on thymidine incorporation between CsA and FK506, despite that FK506 is a much more potent immunosuppressant than CsA.

Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells.

The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl-normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose-dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross-linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.

Chemosensitivity testing of primary cultures of Merkel cell cancer.

Twenty-seven tumor specimens from patients with Merkel cell carcinoma (MCC) were tested for chemosensitivity against a battery of nine cytotoxic drugs in a short-term antimetabolic assay measuring inhibition of thymidine incorporation. Dose-response curves were constructed by plotting drug concentration in micrograms/ml versus % control [3H]thymidine incorporation. Specimens were considered 'sensitive' to a drug if, at the approximate peak plasma concentration (PPC), the inhibition of [3H]thymidine was greater than 50% when compared with untreated control primary cultures. The assay revealed a 'sensitive' tumor in 19 of 20 specimens and 16 of 17 patients had a tumor that was 'sensitive' to at least one drug tested in the assay system. The highest sensitivity in order of frequency was found with doxorubicin, epirubicin, cyclophosphamide, etoposide and cisplatin. At least 40% of the tumors were 'sensitive' to these five drugs. Cyclophosphamide was chosen as the most active drug (at PPC) in 10 of 19 assays (53%), etoposide in seven of 17 (41%), doxorubicin in four of 19 (21%), chlorambucil in one of 12 (8%) and cisplatin in one of 18 (5%) of assays. Though our results are preliminary, we have identified for the first time a range of cytotoxic drugs which appear effective against MCC in vitro. Our main task now is to determine whether our in vitro predictive assay will correlate with clinical benefit to the patient.

Inhibition of Proliferative Activity of Pulmonary Fibroblasts by Tetrandrine

Tetrandrine, an herbal drug, has been employed in China to treat pulmonary fibrosis. To date, the mechanisms governing the antifibrotic action of tetrandrine are unknown. The present study employs a fibroblast mitogenic assay to determine whether tetrandrine directly inhibits the ability of fibroblasts to respond to stimulation by growth factors. The data indicate that tetrandrine blocks proliferation and the incorporation of tritiated thymidine into DNA by fibroblasts stimulated with human serum, PDGF plus plasma, FGF plus plasma, or TNF plus plasma. Since tetrandrine inhibits the response to a variety of growth factors, its action does not appear to involve the blockade of a specific stimulatory receptor. Tetrandrine is effective in inhibiting thymidine incorporation when added up to 6 hr after stimulation of quiescent cells, suggesting either that tetrandrine does not block the attainment of competence by fibroblasts or that its activity is not limited to blocking the attainment of competence by these cells. Growth factor-induced mitogenesis is also inhibited by nitrendipine, a calcium channel blocker, and by cytochalasin B, a microfilament blocker. However, tetrandrine treatment of fibroblasts neither results in the changes of morphology seen with cytochalasin B nor is limited to the early events of stimulus-response coupling. Therefore, the mechanism of action for tetrandrine is not identical to that for either cytochalasin B or nitrendipine. In summary, these results suggest that the antifibrotic action of tetrandrine may be mediated in part by direct inhibition of fibroblast proliferation normally associated with the development and progression of silicosis.

Isolation of a Dihydrobenzofuran Lignan from South American Dragon's Blood (Croton spp.) as an Inhibitor of Cell Proliferation

Dragon's blood is a red viscous latex extracted from the cortex of various Croton spp. (Euphorbiaceae), most commonly Croton lechleri, Croton draconoides (or Croton palanostigma), and Croton erythrochilus. It is used in South American popular medicine for several purposes, including wound healing. Bioassay-guided fractionation of dragon's blood, using an in vitro test system for the stimulation of human umbilical vein endothelial cells, has resulted in the isolation of a dihydrobenzofuran lignan, 3',4-O-dimethylcedrusin or 4-O-methyldihydrodehydrodiconiferyl alcohol [2-(3',4'-dimethoxyphenyl)-3-hydroxymethyl-2,3-dihydro-7-methoxybenzo furan-5- propan-1-ol] [1] as the biologically active principle. A related compound, 4-O-methylcedrusin [2-(3',4'-dimethoxyphenyl)-3-hydroxymethyl-2,3-dihydro-7-hydroxybenzo furan-5- propan-1-ol] [2], and the alkaloid taspine [3], also isolated from dragon's blood, were not active in the same assay. A cell proliferation assay, measuring the incorporation of tritiated thymidine in endothelial cells, showed that compound 1 did not stimulate cell proliferation, but rather inhibited thymidine incorporation, while protecting cells against degradation in a starvation medium.

Atrial natriuretic peptide inhibits growth of hepatoblastoma (HEP G2) cells by means of activation of clearance receptors.

To investigate whether atrial natriuretic factor regulates the growth of hepatocytes and to determine the receptor subtype involved in such modulation, we studied the effect of atrial natriuretic factor 103-126 and clearance receptor binding analogs of atrial natriuretic factor, (des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126) on growth of human hepatoblastoma cells. Atrial natriuretic factor 103-126 and des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 inhibited thymidine incorporation into human hepatoblastoma cells cultured in the presence of bovine serum albumin and epidermal growth factor but not in cells cultured in bovine serum albumin alone. Moreover, atrial natriuretic factor 103-126, des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126, in a concentration-dependent manner, inhibited thymidine incorporation and cell proliferation. As monitored by the ability of des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 to displace 125I-labeled atrial natriuretic factor, epidermal growth factor increased the expression of cell surface clearance receptors. Epidermal growth factor also transiently increased the cellular content of atrial natriuretic factor clearance receptor messenger RNA without altering the levels of guanylyl cyclase-linked atrial natriuretic factor receptor messenger RNA levels. Maximal increase in atrial natriuretic factor clearance receptor messenger RNA coincided with the maximal increase in des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121-displaceable 125I-atrial natriuretic factor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross talk of dual-signal transduction systems in the regulation of DNA synthesis by parathyroid hormone in osteoblastic osteosarcoma cells.

There has been recent evidence that calcium/protein kinase C (Ca/PKC) messenger system as well as adenylate cyclase are involved in the signal transduction stimulated by PTH. We therefore examined the role of these dual-signal transduction systems and the interaction of these systems in the regulation of DNA synthesis by PTH in the osteoblastic osteosarcoma cells, UMR-106. As recently reported, 10(-4) M Sp-cAMPS, a direct activator of cAMP-dependent protein kinase (PKA), and 10(-4) M dibutyryl-cAMP, as well as hPTH-(1-34), caused the significant inhibition of [3H]thymidine incorporation (TdR). Both A23187 and ionomycin (10(-8)-10(-6) M) inhibited TdR in a dose-dependent manner, with a minimal effective dose at 10(-7) M. Although 10(-6) M phorbol 12-myristate 13-acetate (PMA) caused slight but significant stimulation of TdR by itself, it augmented not only dibutyryl-cAMP- but also Sp-cAMPS-induced inhibition of TdR. On the other hand, 4 alpha-phorbol 12,13-didecanoate, incapable of activating PKC, failed to augment these cAMP analogs-induced effects. Pretreatment with 50 microM H-7, an inhibitor of PKC, not only abolished the PMA-induced augmentation of effect by cAMP analogs but also significantly blocked the PTH-induced inhibitory effect on TdR. Pretreatment with 10(-6) M PMA, which downregulates PKC, significantly inhibited the PTH-induced suppression of TdR. Combined treatment with cAMP analog (dibutyryl-cAMP or Sp-cAMPS) and calcium ionophore (A23187 or ionomycin) caused additive effects on TdR, and PMA used in combination with both cAMP analog and calcium ionophore induced the further inhibition of TdR.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth arrest of the breast cancer cell line, T47D, by TNF alpha; cell cycle specificity and signal transduction.

The effects of tumour necrosis factor-alpha (TNF alpha) on the growth and DNA synthesis of the human breast cell line, T47D, were studied. A dose-dependent, reversible inhibition of thymidine incorporation and cell growth was observed in the range of 0.1 ng ml-1 to 100 ng ml-1 of TNF alpha. Cell viability was not impaired in any of the experiments. Flow-cytometric DNA analysis demonstrated that after 24 h exposure to TNF alpha, T47D cells accumulated in the G1 phase of the cell cycle, and were depleted in the G2/M and S phases, suggesting a block in the progression of the G1/S transition. The involvement of protein kinases (PK) and protein phosphatases in TNF alpha-induced signal transduction was also investigated. A transient and rapid 2-fold increase in total cellular protein kinase C (PKC) activity was detected after 10 min exposure to TNF alpha. To study the role of the observed PKC activation in the cytostatic effect of TNF alpha, T47D cells were exposed to the cytokine in the presence of the potent PKC inhibitor, H7. The inhibitory effect of TNF alpha on thymidine incorporation was not affected by exposure to H7 at concentrations sufficient to block the stimulation of thymidine up-take induced by the PKC agonist, phorbol-12-myristate-13-acetate (PMA). The involvement of other signalling pathways was addressed using the cyclic nucleotide-dependent PK inhibitor, H8; the calmodulin-dependent PK inhibitor, W7; and the inhibitor of protein phosphatases PP1 and PP2B, okadaic acid. Exposure of T47D cells to these enzyme inhibitors failed to antagonise the inhibition of thymidine incorporation by TNF alpha. Taken together, these results indicate that the cytostatic effect of TNF alpha on T47D cells occurs at the G1/S transition of the cell cycle, and is mediated by an intracellular pathway which does not involve the activity of protein kinases C and A, nor protein phosphatases PP1, PP2B.

Structure-Anticancer and Structure-Genetic Activity Relationships of Homo-aza-steroidal Esters of N,N-Bis(2-chloroethyl)aminocinnamic Acid Isomers

Four steroidal lactams of the A- and D-rings were used for the esterification in the C-3 or C-17 positions, respectively, of their nuclei with the W,N-bis(2-chloroethyl)aminocinnamic acid isomers. The con-densation reaction of the hydroxylic group of the steroidal lactams with each mustard was effected in dichloromethane in the presence of the catalyst p-dimethylaminopyridine and dicyclohexylcarbodiimide as de-hydrating agent. The esters were obtained in pure form after column chromatography, and their structures were verified and confirmed by analytical methods (IR and UV spectra). The 12 esters were tested in vivo against P388, L1210 leukemias, Ehrlich ascites tumor, and melanoma B16. The esters 3α-hydroxy-13α-amino-13,17-seco-5α-androstan-17- oic-13,17-lactam-o,m,p-N,N-bis(2-chloroethyl)aminocinnamates, in which the alkylating agents are linked to the modified steroid in the axial position, are inactive in the above experimental animal tumor systems. The effect of the homo-aza-steroidal esters of N,N-bis(2-chloroethyl)ami- nocinnamic acid isomers on the incorporation of radioactive precursors into DNA, RNA, and proteins of L1210, P388 leukemias, Ehrlich ascites tumor, and baby hamster kidney cells was investigated. Higher inhibitory effects on the incorporation of the radioactive precursors was obtained with the ortho-derivatives, yielding >40% inhibition of thymidine incor-poration in all tumor lines tested. The effect of four esters in which the m-N,N-bis(2-chloroethyl)aminocinnamic acid is linked to the modified steroids on sister chromatid exchanges in human lymphocyte culture was investigated.

Beta-endorphin is a potent inhibitor of thymidine incorporation into DNA via mu- and kappa-opioid receptors in fetal rat brain cell aggregates in culture.

Thymidine incorporation into DNA was inhibited dose-dependently by beta-endorphin in rat fetal brain cell aggregate cultures. The inhibition was reversed partially by mu (cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr- Pen-Thr amide) or kappa (norbinaltorphimine) antagonists. Complete blockade of the beta-endorphin inhibitory effect was achieved only on concomitant exposure to both antagonists. Eadie-Hofstee analysis revealed that beta-endorphin inhibited thymidine incorporation noncompetitively. In the presence of protease inhibitors, beta-endorphin decreased thymidine incorporation with an IC50 of 0.7 nM. Truncated and N-acetylated beta-endorphin derivatives, which bind with low affinity to opioid receptors, did not affect thymidine incorporation. These findings indicate that beta-endorphin at physiological concentrations can regulate thymidine incorporation in cultured brain cells.

Effect of 1.25-Dihydroxyvitamin D3 on the Proliferation of Osteoblastic MC3T3-E1 Cells by Modulating the Release of Local Regulators from Monocytes

Some evidence suggests an important role of mononuclear cells at the bone remodeling sites in the coupling of bone resorption to bone formation. Therefore, we examined effects of human monocytes-conditioned medium (CM) treated with 1.25-dihydroxyvitamin D3 [1,25 (OH) 2D3] on the proliferation of osteoblastic MC3T3-E1 cells. 1,25 (OH) 2D3 (10−11-10−8) directly inhibited (3H) thymidine incorporation(TdR) into MC3T3-El cells in a dose-related fashion. 24,25 (OH) 2D3 and 25 (OH) D3 (10−8 and 10−7 M) had only modest inhibitory effect, compared to that of 1,25 (OH) 2D3. CM, per se, on the other hand, significantly stimulated TdR, whereas CM obtained from monocytes treated with 1,25 (OH) 2D3 (10−10 and 10−8 M) significantly inhibited TdR. Treatment of monocytes with 10−7 M 25 (OH) D3 significantly inhibited CM-induced stimulation of TdR to a lesser degree, compared to that of 10−8 M 1,25 (OH) 2D3 and treatment of monocytes with 10−7 M 24,25 (OH) 2D3 did not affect CM-induced stimulation of TdR. The inhibition of TdR by 1,25 (OH) 2D3-treated CM was significantly blocked by 10−6 M indomethacin, an inhibitor of prostaglandin synthesis. Present data first indicate that 1,25 (OH) 2D3 inhibited osteoblast proliferation not only directly but also indirectly through modulating the release of local factors as to bone remodeling from monocytes, and also suggest that its indirect effect via monocytes was mediated at least in part through prostaglandin synthesis.