Mitogenic Substances Research Articles

Abstract A16: A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma

Abstract Background: Pancreatic cancer (PaCa) is a leading cause of cancer-related deaths and is projected to be the second most common cancer in a decade. It is characterized by a dense stroma surrounding tumor cells, known as desmoplasia that is stimulated by Sonic Hedgehog (SHH) released by PaCa cells. Stromal cells, in turn, release mitogenic substances such as hepatocyte growth factor (HGF) and Insulin-like growth factor receptor (IGF-1) by glioma-associated oncogene homolog 1 (GLI1) transcription factor that stimulate tumor growth, invasion, and resistance to therapy. We investigated the potential of targeting the metastasis suppressor gene N-myc downstream regulated gene 1 (NDRG1) to interrupt the oncogenic crosstalk between cancer associated human pancreatic stellate cells (PSCs) and PaCa cells. Methods: We assessed novel thiosemicarbazone anti-cancer agents, Dp44mT and DpC, that upregulate NDRG1 and their effects on: (i) SHH production by PaCa cells and also HGF and IGF-1 production by PSCs; (ii) HGF and IGF-1 receptors and their downstream signaling in indirect 2D and direct 3D co-culture of PSCs and PaCa cells; (iii) GLI1 and its upstream mediators in PSCs; and (iv) proliferative and metastatic properties of PaCa cells co-cultured with PSCs. Results: Dp44mT and DpC upregulated NDRG1 and reduced SHH production by PaCa. These agents also inhibited downstream HGF and IGF-1 signaling pathways in PaCa cells, leading to reduced metastatic potential in co-culture 2D and 3D models. Further, these agents also inhibited GLI1 levels and activation in PSCs, leading to reduced HGF and IGF-1 production by these stromal cells. NDRG1 overexpression alone had similar effects, while silencing NDRG1 further potentiated the oncogenic crosstalk between PaCa and PSC cells. Conclusions: Novel agents targeting NDRG1 have the unique ability to block the oncogenic crosstalk between PaCa cells and the surrounding stroma. This presents a novel approach to PaCa treatment that can potentially overcome the major problems attributed to desmoplasia, including therapeutic resistance and metastasis. Citation Format: Bekesho Geleta, Kyung Chan Park, Patric J. Jansson, Minoti Apte, Des R. Richardson, Zaklina Kovacevic. A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A16.

Peripheral Blood Lymphocyte and Platelet STEM CELL Expansion in Autologous Platelet-Derived Growth Factor-RICH Plasma

AbstractAbstract 4717Research on stimulatory and inhibitory factors present in the hematopoietic microenvironment promoted development of ex-vivo cell culture methods. Recent investigations strive to select natural and novel mitogenic substances within defined conditions to culture and harvest specific progenitor cells and cell lines. Optimal large scale cell expansion of HSCs remains to be developed. The present study documents ex-vivo human lymphocytes and platelet culture in autologous human blood plasma (BP) complemented with a novel ionic metal polysulphide. Resulting plasma is growth-factor rich (FRP) with a high platelet count (HPC) containing > 4.5×10E6 plts/ul. Co-culture expansion for 3-days of normal peripheral blood cells from n=20 healthy individuals in FRP promotes lymphoid and platelet series stem cell lineage growth and differentiation. Cultured cells characterized using conventional hematology and flow cytometry methods on CD3, CD4, CD8, CD34, CD38, CD41, CD61 antigen bearing cells; and average values reported. Result analysis of CD3 and CD4 bearing cells before-after a 3-day culture indicates a 4 % and 18 % respective increase, while CD8 cell population decrease 7%. A stimulation response obtained on CD34 and CD38 cells with a 67.30% and 53.89 % respective increase. Cell growth of CD41 and CD61 antigen bearing cells increased 50.89 % and 69.22 %, respectively. Functional platelet counts (before-after culture) correspond with a 3–5 fold increase of original concentration, and presence of recently reported proto-platelets. Minimum and maximum values varied among studied population. Discrete elevation in Neutrophils and Granulocytes counts observed. Potential applications thought for immunosuppression, cancer and organ transplant. Disclosures:No relevant conflicts of interest to declare.

Secreted Opisthorchis viverrini glutathione S-transferase regulates cell proliferation through AKT and ERK pathways in cholangiocarcinoma

Opisthorchis viverrini can develop mitogenic substances into the excretory/secretory product (ESP) that may play an important role in promoting the genesis of cholangiocarcinoma (CCA). In the present study, glutathione S-transferase (GST) is identified as being secreted into Ov-ESP and acting as one of the parasitic mitogens. Its proliferative effect and possible mechanism were explored and its association with the tumor development is proposed. Ov-ESP was concentrated and purified by gel filtration chromatography. SDS-PAGE, 2-DE, and LC-MS/MS identified GST predominantly expressed in the proliferative ESP fraction. The recombinant OvGST (r OvGST) was produced by wheat germ cell-free expression and confirmed by an MTS assay to have a proliferative function on NIH-3T3 murine fibroblasts and MMNK1 non-tumorigenic human bile duct epithelial cells in a dose dependent manner with different optimal doses. The cell surface binding of r OvGST was confirmed in vitro and the activation of both pAKT and pERK was revealed as the mechanism of OvGST-mediated cell proliferation. With support from the observation of secreted OvGST on the biliary cells surrounding the parasites, it is suggested that OvGST can promote cell proliferation that consequently may accelerate the genesis of CCA.

The complexity of platelet metabolism and its contribution to atherothrombosis

Platelet functions are multiple, complex and not limited to haemostasis. In fact, platelets play a relevant role in vascular inflammation and atherosclerosis (ATS). In the presence of vascular lesions or inflammation, endothelial denudation or activation triggers mechanisms that render the circulating platelets adhesive for the vascular wall. Endothelial lesions expose subendothelial matrix components, such as collagen, von Willebrand factor, fibronectin and other adhesive proteins. Platelet adhesion depends on the interaction between these components and platelet receptors (mainly glycoprotein (GP) VI and GPIb-IX-V). Adhesion triggers the platelet release of inflammatory and mitogenic substances that alter the thromboresistant endothelial surface, enhance the chemoattraction of leukocytes, stimulate smooth muscle cell proliferation and contribute to matrix degradation. Finally, GPIIb- IIIa receptors are activated, leading to firm platelet aggregation and thrombus formation. Platelets participate in the formation of mural thrombi in the late stages of atherosclerotic disease, but also adhere to endothelial cells during the earlier stages of atherosclerotic plaque development. Moreover, platelets exert important functions in modulating inflammatory and immune processes. An improved comprehension of the complex platelet pathophysiology could suggest new therapeutic strategies to reduce the impact of atherosclerotic disease.

SCUBE1 — A new scoop in vascular biology?

See article by Tu et al. [6] (pages 486–495) in this issue. Platelets represent an important link between inflammation, thrombosis, and vascular and tissue repair mechanisms. Once activated, platelets generate and release an arsenal of potent inflammatory, proliferative, and mitogenic substances into the local microenvironment of vascular lesions [1]. Thereby, platelets control and effect multiple mechanisms of cellular function involved in vascular and tissue repair. The epidermal growth factor (EGF) superfamily is a group of growth factors, cytokine-like mediators, and extracellular matrix proteins. A new gene of an EGF-related protein was isolated 6 years ago in developing mice [2]. This new mammalian gene encodes a protein with a signal peptide at the amino-terminus followed by several EGF-like repeats and one CUB-domain at the carboxyl terminus. This gene family was termed SCUBE for s ignal-peptide- CUB - E GF-like domain-containing proteins. … * Corresponding authors. Tel.: +49 7071 29 83688; fax: +49 7071 29 5749. Email address: stephan.lindemann{at}med.uni-tuebingen.de meinrad.gawaz{at}med.uni-tuebingen.de

Cyclooxygenase-2 (COX-2) enzyme inhibitors and radiotherapy: preclinical basis.

Cyclooxygenase-2 (COX-2), an enzyme induced by proinflammatory cytokines, mitogenic substances, oncogenes, growth factors, and hypoxia, among others, is involved in the metabolic conversion of arachidonic acid to prostaglandins in inflamed tissues and neoplasia. COX-2 is often overexpressed in malignant tumors and premalignant lesions and is linked to carcinogenesis, maintenance of progressive tumor growth, and facilitation of metastatic spread. Because COX-2 may also be a determinant of tumor radioresistance, its inhibition or inhibition of its products (prostaglandins) may improve tumor response to radiotherapy. Preclinical studies have shown that treatment with selective COX-2 inhibitors significantly enhances tumor response to radiation without appreciably affecting normal tissue radioresponse. The underlying mechanisms of the COX-2 inhibitor-radiation interactions seem to be multiple, with the enzyme inhibitor directly or indirectly augmenting tumor cell destruction by radiation. Thus, use of selective COX-2 inhibitors is a potential approach for improving cancer radiotherapy.

Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Microculture tetrazolium assay (MTT) application for measuring cell-mediated immunity (CMI) and evaluating certain mitogenic substances as future drugs of immune therapy for head and neck cancer patients (HNCA)

Microculture tetrazolium assay (MTT) application for measuring cell-mediated immunity (CMI) and evaluating certain mitogenic substances as future drugs of immune therapy for head and neck cancer patients (HNCA)

Trypanosoma cruzi mitochondrial malate dehydrogenase triggers polyclonal B-cell activation.

It has been proposed that Trypanosoma cruzi, the aetiologic agent of Chagas' disease, produces mitogenic substances responsible for the polyclonal B-cell activation observed during the acute phase of the infection. Isolation and characterization of the molecules involved in the induction of polyclonal activation observed during infectious diseases have posed a great challenge for the immunologist over the last decade. In this work we report that a 33 kD protein obtained from an alkaline fraction of T. cruzi epimastigotes (FI) stimulates proliferation and promotes differentiation into antibody-secreting cells of normal murine B cells in a T-cell independent manner. By flow cytometry we also found that the 33 kDa protein induces an increase in the expression of MHC class II and B7.2 but not B7.1 molecules on the B-cell surface. Sequencing by mass spectrometry identified the T. cruzi 33 kD protein as hypothetical oxidoreductase, a member of the aldo/ketoreductase family. In this report we demonstrate that this protein is also present in the infective bloodstream trypomastigote form of the parasite and was identified as T. cruzi mitochondrial malate dehydrogenase (mMDH) by enzyme activity and by Western blotting using a specific mMDH polyclonal antiserum. The biologic relevance of mMDH-induced polyclonal activation concerning T. cruzi infection is discussed.

The possible association of in vivo leukocyte-platelet heterophilic aggregate formation and the development of diabetic angiopathy

Circulating leukocyte-platelet heterophilic aggregates produce procoagulant, oxidative and mitogenic substances, and can cause microembolism in capillaries as well as acute arterial thrombosis. Our aim was to determine if there was any difference in the number of circulating heterophilic aggregates between diabetic patients and controls, if the formation of aggregates correlated with the actual HgbA1c level, duration of diabetes and postprandial rise in serum glucose level, with different vascular complications and whether decreasing postprandial serum glucose had any effect on heterophilic aggregate formation. The number of circulating heterophilic aggregates was measured in 90 diabetic patients (Type 1, 29; Type 2, 61) and in 23 control subjects by a flow-cytometric assay, and the result was given as percentage of the respective leukocyte subsets. There was no significant difference in lymphocyte-platelet and neutrophil-platelet aggregate number in patients and controls; however, there was a significant difference in the percentage of monocyte-platelet aggregates between the diabetic and control group (Type 1, 43.0 - 17.8; Type 2, 34.9 - 12.5; control, 24.6 - 8.2; P < 0.01 and P < 0.5, respectively). Patients with proliferative retinopathy and nephropathy showed the highest number of monocyte-platelet aggregates. No significant correlation was, however, found with HgbA1c. In Type 2 diabetes a non-significant, but remarkable, tendency between elevation of postprandial serum glucose levels and platelet-monocyte aggregate formation was observed and acarbose seemed to be effective in decreasing both. This study provides further support that heterophilic aggregates might have role in the pathogenesis of diabetic vascular complications.

Pasteurella multocida Toxin Stimulates Mitogen-activated Protein Kinase via Gq/11-dependent Transactivation of the Epidermal Growth Factor Receptor

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.

Effect of linoleic acid supplements on vessel wall hyperplasia in rabbits.

Intimai hyperplasia is a major complication of vascular surgical procedures.1–5 In addition, intimai hyperplasia contributes significantly to the development of atherosclerosis.6 Hyperplasia is a result of platelet/leukocyte/injured vessel wall interactions, followed by the release of vasoactive, chemotactic and mitogenic substances which promote smooth muscle cells migration and proliferation.6, 7 Fatty acid metabolites synthesized by vascular wall and blood cells are thought to modulate, in part, the onset of intimai hyperplasia and atherosclerosis. For example, 13-hydroxyoctadecadienoic acid (13-HODE), derived from linoleic acid via the lipoxygenase pathway, has been shown to inhibit platelet adhesion to the subendothelial layer.8, 9 Since platelet adhesion to the vessel wall is a prerequisite to the secretion of platelet-derived smooth muscle cell, and linoleic acid is metabolized into 13-HODE the vessel wall, we determined whether a linoleic acid-rich diet, given before injury, was associated with decreased intimal hyperplasia following vessel wall balloon catheter injury in normo- and hypercholesterolemic rabbits.

Analysis of mitogenic substances in Bupleurum chinense by ESR spectroscopy.

The polyphenolic substance(s) in the hot water extract of Bupleurum chinense (PSF) showed strong mitogenic activity. In this paper, we analyzed PSF by using ESR spectroscopy, and found that i) PSF showed a strong ESR signal on g = 2.005 which was similar to the commercially available lignin; ii) Sho-saiko-to, which contains an extract of B. chinense, also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on g = 2.005. Peroxidase activity, essential for producing polyphenolic substances, was detected in the cold water extract of B. chinense. In addition, the signal intensity of the ESR spectrum of B. chinense was increased after boiling. The data of the ESR spectra of the model reactions using lignin, arginine, proline and maltose also strongly suggested that a certain chemical modification proceeded during the hot water extraction to increase the percentage of the stable free radical. These facts strongly suggested that the mitogenic substance in B. chinense is a polyphenolic substance extracted by hot water, and the structure was modified during the extraction to increase the stable free radical components.

Megakaryocytes and Platelets in Diabetes Mellitus

SummaryIn patients with diabetes mellitus vascular diseases and related complications still represent the main cause of death. Neuropathy, nephropathy, retinopathy and disturbed nutritive tissue perfusion result from reduced capillary microcirculation. Macroangiopathic mechanisms lead to accelerated cerebral, coronary and peripheral artery disease precipitating life terminating thrombotic events superimposed to those vascular lesions. Thus morbidity and mortality depend mainly on vascular complications. The functional thromboresistance is reduced in the diabetic state with hyperactive platelets operating by three mechanisms: 1) microembol-isation of the capillaries; 2) local progression of preexisting vascular lesions by secretion of constrictive, mitogenic and oxidative substances; 3) trigger of the prognosis limiting arterial thrombotic event. The increased functional properties of di-abetic platelets result from the primary release of larger platelets with enhanced thromboxane formation capacity and increased numbers of functional glycoprotein receptors GPIb and GPIIb-llla which are synthesized in the megakaryocytes. It could be demonstrated with the Düsseldorf III method of flowcytometric activation marker testing (CD62, CD63, thrombospondin) that predominantly large platelets circulate in an activated state even in very early (pre)diabetic stages sug-gesting that the megakaryocyte-platelet system might be systemically affected. Based on these observation a "stem cell hypothesis" is developed which suggests that bone marrow derived platelets may be systemically involved in the pathogenesis of the vascular complications and eventually of type-l diabetes. The idea of antiplatelet agents as an useful early adjunct to metabolic therapy is promoted.

Oxidized LDL Induces Transcription Factor Activator Protein–1 but Inhibits Activation of Nuclear Factor–κB in Human Vascular Smooth Muscle Cells

Oxidized LDL (Ox-LDL) has been implicated in the development of atherosclerotic lesions, mainly due to its enhanced uptake by macrophages and its ability to alter gene expression in arterial cells. In the present study we demonstrated that Ox-LDL activates activator protein-1 (AP-1), a transcription factor generally induced by mitogenic substances. Lysophosphatidylcholine, which is generated during oxidation of LDL, stimulated AP-1 in a dose-dependent manner. In contrast, the radical-dependent transcription factor nuclear factor-kappa B (NF-kappa B) was not activated by Ox-LDL, and at a concentration of 50 micrograms/mL, Ox-LDL inhibited lipopolysaccharide-induced activation of NF-kappa B. Oxysterols but not lysophosphatidylcholine inhibited lipopolysaccharide-induced NF-kappa B activation, suggesting that they may be responsible for the inhibitory effect of Ox-LDL. In conclusion, Ox-LDL has opposing effects on the activities of NF-kappa B and AP-1, suggesting involvement of mechanisms for transcriptional regulation that are strongly affected by lipid oxidation products.

Fractionation and characterization of anticomplementary and mitogenic substances from Panax ginseng extract G‐115

AbstractPanax ginseng extract G‐115 had anticomplementary and mitogenic activities. Incubation with artificial gastric juice slightly decreased the anticomplementary activity, whereas incubation with artificial intestinal juice or with artificial gastric juice followed by artificial intestinal juice slightly increased the activity. Incubations with artificial gastric and intestinal juices slightly decreased mitogenic activity similar to that with water. The G‐115 was fractionated in order to characterize the active substances for anticomplementary and mitogenic activities. The most potent anticomplementary activity was observed in the crude polysaccharide fraction, G‐115G, whereas the water‐soluble dialysable fraction, G‐115E showed the most potent mitogenic activity. Although pronase digestion did not change the anticomplementary activity of G‐115G, G‐115G decreased the anticomplementary activity after the treatments with periodate and endo‐polygalacturonase indicating that a carbohydrate moiety containing polygalacturonan may contribute to the activity. G‐115G was further purified by precipitation with cetyltrimethylammonium bromide, anion‐exchange chromatography on DEAE‐Sepharose and gel filtration on Sepharose CL‐4B, and major potent anticomplementary polysaccharide, G‐115I1‐IIa‐2‐3 was obtained. G‐115I1‐IIa‐2‐3 was homogeneous, and its molecular weight was estimated to be 3.68 × 105. G‐115I1‐IIa‐2‐3 consisted mainly of arabinose, galactose and glucose in addition to small amounts of galacturonic acid, glucuronic acid and rhamnose.

Characterization of mitogenic substances in the hot water extracts of bupleuri radix.

Bupleuri Radix is a commonly used medicinal plant in Kampo medicine, and its hot water extracts show mitogenic activity to murine lymphocytes. In this paper the mitogenic substances in the hot water extracts of Bupleuri Radix (Bup-HWE) were fractionated and characterized physicochemically and immunologically. Most of these substances were recovered from mol. wt of more than 200 kDA fraction (fr. C-13). Separation of fr. C-13 by phenol-water fractionation method gave water soluble and phenol soluble mitogenic substances. These substances showed the activity even in C3H/HeJ mice, and polymyxin B or lysozyme treatment did not abrogate the activity, suggesting that the active substances are not related to bacterial lipopolysaccharide. Treatment of the mitogenic substances recovered from the phenol layer with NaCLO2, a polyphenol degrading chemical, significantly reduced the activity, but pronase and pectinase treatments were not effective. The mitogenic substances in the water layer were active even after NaCLO2 treatment. These findings suggested that the mitogenic substances of Bup-HWE are large molecular weight polyphenolic compounds and polysaccharide. The mitogenic substances are suggested to be B cell mitogens.

ADP causes partial degranulation of platelets in the absence of aggregation.

Whole blood flow cytometry has revealed that platelets undergo partial degranulation in response to ADP, in the absence of aggregation, as evidenced by the expression of the P-selectin and CD63 antigens of the alpha-granule and lysosomal membranes respectively. With maximum ADP (10(-5) M) fibrinogen bound to 76.1 +/- 7.2% of platelets but P-selectin and CD63 antigen were expressed on 26.9 +/- 9.8% and 8.6 +/- 3.5% of platelets respectively. Maximum fibrinogen binding, P-selectin and CD63 expression induced by alpha-thrombin were 96.1 +/- 1.4%, 92.8 +/- 2.3% and 77.6 +/- 9.7% respectively. beta-thromboglobulin release from the ADP-stimulated platelets correlated closely with the expression of P-selectin and CD63 (r = 0.98 +/- 0.02 for both antigens). No platelet aggregates were seen by flow cytometry and the absence of aggregation was confirmed by single cell counting. Addition of the GPIIb-IIIa antagonist echistatin, at concentrations that totally blocked fibrinogen binding to ADP-stimulated platelets, had no effect on the expression of the granule membrane antigens. The partial degranulation of normal platelets was independent of thrombin generation since it was not inhibited by hirudin (5 units/ml). In conclusion, ADP is capable of causing partial degranulation of platelets independently of aggregation, fibrinogen binding or thrombin generation. Thus release of potent procoagulant, vasoactive and mitogenic substances from the platelets could continue in the presence of thrombin inhibitors and GPIIb-IIIa antagonists.

Anti-proliferative effects of deflazacort on NB2 cells as quantitated by formazan production

Prolactin and other lactogenic hormones are mitogenic for the rat T-cell lymphoma line, Nb2. Glucocorticoids have antiproliferative effects on these cells. A limiting feature of experiments utilizing the Nb2 line is their labor-intensive nature. We therefore adapted the commonly used MTT dye proliferation assay for the Nb2 cell line. While rPRL, hPRL, oPRL, hGH, bPL, and to a lesser extent bPRL stimulated the Nb2 cells, hormones without lactogenic activity, rGH and oGH did not. Human serum and rat sera from animals bearing a PRL-secreting tumor stimulated the Nb2 cells in parallel to standards. Glucocorticoids had anti-proliferative effects on Nb2 cells in the presence of half-maximal or maximal PRL doses, as measured by the MTT proliferation assay. It has been claimed that an oxazoline steroid, deflazacort, has anti-inflammatory effects in clinical studies with fewer of the deleterious side-effects common to glucocorticoids. We therefore compared the in vitro anti-proliferative effects of deflazacort with other glucocorticoids. Deflazacort's negative effect on Nb2 cell proliferation was similar to that of cortisol and prednisolone and less than that of dexamethasone. We conclude that the MTT proliferation assay can be used to study both mitogenic and anti-proliferative substances in Nb2 cells. In addition we found that deflazacort acts similarly in vitro to other glucocorticoids.

Molecular biology of intimal proliferation

Arterial intimal proliferation occurs in response to acute and chronic injuries such as angioplasty, surgical trauma, immune injury, and hyperlipidemia. Although there are subtle differences, all lesions contain smooth muscle cells, macrophages, and occasional lymphocytes imbedded in an extracellular matrix. The vascular injury elicits a cascade of chemotactic and mitogenic substances that augment and induce gene expression of growth factors such as platelet-derived growth factor, insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, and their receptors. Binding of the mitogenic polypeptide growth factors to their specific cell surface receptor stimulates intracellular messengers such as tyrosine kinases, cyclic AMP, protein kinase C, phosphoinositol, and calcium, resulting in expression of oncogenes like c-fos, c-jun, c-myc and c-myb. Although the molecular mechanisms for intimal smooth muscle cell proliferation are far from elucidated, the increasing understanding of the molecular mechanisms involved has enabled the demonstration of inhibition of myointimal hyperplasia in animal models by antisense oligonucleotides and experimental gene transfer.