Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.     

Extracellular signal-regulated kinase (MAP kinase) immunoreactivity in the rhesus monkey brain

Phosphorylation of both tyrosine and serine residues of focal adhesion kinase (FAK) was stimulated by the adhesion of BALB/c mouse 3T3 cells to fibronectin, but phosphorylation of threonine was not detectable. Acidic and basic fibroblast growth factors also stimulated the phosphorylation of serine and tyrosine of FAK in cells adhered to poly-L-lysine, but epidermal growth factor and platelet-derived growth factor did not. A fusion protein of fibronectin and basic fibroblast growth factor effectively induced the phosphorylation of FAK. Phosphorylation of FAK in the rat myoblast L-6 cell line, which lacks fibroblast growth factor receptors, was not stimulated by fibroblast growth factors, suggesting that the interaction of fibroblast growth factors with their receptors might cause the phosphorylation of FAK.     

Small intestinal submucosa: a substrate for in vitro cell growth

The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and the external muscular layers. The remaining 80 microns thick sheet was disinfected and sterilized by methods which removed all cellular components. The SIS-ECM, retaining its native 3-dimensional microarchitecture and composition, was evaluated for its ability to support in vitro cell growth. Six separate cell types were seeded either alone or in coculture with other cells upon this matrix, grown in selected media, a examined daily for time periods ranging from 48 h to 2 weeks. The six cell types tested were NIH Swiss mouse 3T3 fibroblast, NIH 3T3/j2 fibroblasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteosarcoma (ROS) cell line. All cell types showed the ability to attach a proliferate. All fibroblast cell line and the keratinocytes proliferated and/or migrated into the 3-dimensional scaffold of the SIS matrix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts and primary human keratinocytes resulted in a distinctive spatial orientation of the two cell types. The fibroblast populated the mid-substance of the 3-dimensional matrix and the keratinocytes formed an epidermal structure with rete ridge-like formation and stratification when the composite was lifted to an air liquid interface in culture. In summary, SIS provides a substratum with a 3-dimensional scaffold that allows for cell migration and spatial organization. The substratum is suitable for in vitro studies of the interaction between epithelial or mesenchymal cells and a naturally occurring extracellular matrix.     

Inhibition of natural killer cell cytotoxicity by cell growth-related molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

Spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.     

Body/self awareness and interpersonal communications: fundamental components of reproductive health awareness

We investigated the role of Sonic hedgehog (SHH) in osteoblast differentiation and bone formation. The numbers of ALP-positive cells in the mouse fibroblastic cell line C3H10T1/2 and the mouse osteoblastic cell line MC3T3-E1 were increased by co-culture with chicken fibroblasts transfected with chicken Shh cDNA encoding amino-terminal peptide (Shh-N). The conditioned medium of Shh-N-RCAS-transfected chicken fibroblast cultures also significantly increased ALP activity in both C3H10T1/2 and MC3T3-E1 cells. Intramuscular transplantation of Shh-N-RCAS-transfected chicken fibroblasts into athymic mice induced ectopic bone formation. These results indicate that SHH induces osteoblast differentiation and ectopic bone formation.     

The regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the isolated perfused rat liver

The effect of perfusion of an isolated rat liver on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was studied. In liver removed during the basal period of the diurnal cycle of enzyme activity, a 227 +/- 41% increase in enzyme activity occurred after 3 h of a plasma-free perfusion. This could be prevented by the addition of cycloheximide or pure cholesterol (dispersed with lecithin) to the perfusate. In contrast, the continuous addition of taurocholate or taurochenodeoxycholate, alone or in combination, at a variety of rates did not prevent the increase in enzyme activity. The added bile salts were efficiently extracted from the perfusate and excreted in the bile. The addition of these bile salts to a cholesterol-enriched perfusate did not alter the effect obtained with cholesterol alone. If the perfusate contained whole serum, the increase induced by perfusion in the basal period was smaller (88 +/- 27%) than with plasma-free perfusate. Again, the major bile salts of the rat failed to prevent the increase in enzyme activity induced by liver perfusion. If livers were removed and perfused at the height of the diurnal cycle of enzyme activity, the enzyme activity remained high (2 +/- 10% increase) rather than decreasing, as occurs in vivo. If cholesterol was added to these perfusions, a 52 +/- 4% decrease was induced. Bile salt addition induced no decrease. From the results it is concluded that the major bile salts are not direct regulators of hepatic cholesterol synthesis, but pure cholesterol, in the absence of bile salt or lipoprotein, is able to initiate the mechanism that represses hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.     

Hemoregulatory peptide (HP5b) dimer effects on normal and malignant cells in culture

The dimer of the hemoregulatory peptide HP5b has been investigated for biological effects on various cell types in culture including mouse granulocyte-macrophage colony forming units (CFU-GM) from agar and murine long-term bone marrow culture (LTBMC). While CFU-GM were significantly stimulated in both systems, mitogen activation of mouse T, B and natural killer (NK) cells was not affected. Peptide treated mouse 3T3 fibroblasts reached a higher saturation density than controls; otherwise no effect was seen. A series of malignant cell lines was also tested. On a human glioblastoma cell line (GaMg) and rat glioma cell line (BT5C) a slight but significant stimulatory effect was found, while human mammary carcinoma cells (MCF7) were not affected. On SC1 mouse lymphoma cells a slight stimulation of cell growth was seen during the first part of exponential growth. Since HP5b acts as a stimulator for stromal cell secretion of other growth factors, supernatants from a human bone marrow stromal cell line stimulated with HP5b were tested on various cell lines. The effects of the supernatants on cell growth of the tested cell lines were not affected by HP5b treatment. Taken together with available in vivo data, the results indicate that the hemoregulatory peptide is a selective stimulator of myelopoiesis.     

Cloned 3T6 cell line from CD-1 mouse fetal molar dental papillae

Only primary pulpal cell cultures and one virally transformed mouse cell culture have been formally reported in the literature to synthesize proteins such as phosphophoryn which are unique to dentin matrix. In the present study, a mixed culture was derived from dental papilla cells of 18-19 fetal day CD-1 mouse mandibular first molars, maintained on a 3T6 plating regimen, and subsequently cloned after 28 passages. This cloned cell line (MDPC-23) exhibited several unique features, some of which were characteristic of odontoblasts in vivo. The features of this cell line included (1) epithelioid morphology of all cells with multiple cell membrane processes, (2) high alkaline phosphatase activity in all cells, (3) formation of multilayered nodules and multilayered cultures when maintained in ascorbic acid and beta-glycerophosphate, and (4) expression of two markers for odontoblast differentiation, i.e. dentin phosphoprotein and dentin sialoprotein.     

Interleukin 12 augments the liver-associated immunity and reduces liver metastases

BACKGROUND/AIMS: It has been reported recently that in vivo administration of interleukin-12 (IL-12) augments the cytotoxic activity of natural killer (NK)/T cells and shows a powerful anti-tumor activity. In this study, we evaluated that the IL-12 effect on liver-associated immunity and in vivo efficacy on the hepatic metastases in a rat model. MATERIALS AND METHODS: Varying amounts of mouse recombinant IL-12 were injected intraperitoneally for 5 days to adult male Fischer rats and hepatic sinusoidal lymphocytes (HSL) were collected. Purified HSL are spontaneously cytolytic to both conventional NK-sensitive target (YAC-1) and NK-resistant target (RCN-H4) tumor cells. RESULTS: IL-12 was found to increase the number of HSL and the cytolytic activity against these target cells in a dose-dependent fashion. Flow cytometric analysis showed that IL-12 caused an increase of CD8+ subpopulation in HSL and a double staining study revealed that the increased subpopulation was not CD3+8+ (cytotoxic T cell) fraction, but actually CD3-8+ (NK cell) fraction. Experimental liver metastases was markedly reduced in rats treated intraperitoneally with IL-12. CONCLUSION: These results demonstrate that IL-12 augments the cytolytic activity of HSL and suggests this cytokine as an attractive choice for liver metastases therapy.     

Spontaneous and asbestos-induced transformation of mesothelial cells in vitro

The processes of spontaneous and asbestos-induced transformation of rat mesothelium were studied using cell cultures obtained in the laboratory. The same changes in cell properties were established in both spontaneous and asbestos-induced transformation: change in epidermal growth factor (EGF) response, in some cases appearance of fibroblast-like cells instead of polygonal ones, appearance of multilayer cell growth foci, and ability to grow in semisolid agar. The response to fibroblast growth factor, insulin-like growth factor 1, and insulin did not change during transformation as well as the P450 system activity measured by benz(a)pyrene (BP) and 7,12-dimethylbenzanthracene (DMBA) cytotoxicity. The asbestos-induced transformation began earlier than the spontaneous one. EGF began to stimulate mesothelium proliferation instead of its inhibition at 6-7 passages in the case of asbestos-induced transformation, whereas during spontaneous transformation this change began at 9-10 passages. Elongated rather than polygonal cells appeared at 10-11 instead of 17-18 passages (this morphological change did not take place at all lines studied). The ability to grow in semisolid agar was found at 14-16 passages with asbestos and at 22-24 passages without it. The results allow us to propose the necessity of a positive EGF response for mesothelial cell transformation and the similarity of mechanisms of spontaneous and asbestos-induced transformation.     

Blocked negative selection of developing T cells in mice expressing the baculovirus p35 caspase inhibitor

Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).     

Osteogenic protein-1 protects against cerebral infarction induced by MCA ligation in adult rats

A series of studies using farnesyltransferase (FTase) inhibitors that the inhibition of FTase function suppresses the growth of ras-transformed cells in vitro and in vivo. However, whether FTase is directly involved in the regulation of cell proliferation remains to be demonstrated. To investigate whether overexpression of FTase results in altered cell growth and transformation, we thus used NIH3T3 cells transfected with cDNA constructs of both alpha and beta subunits of human FTase. FTase-overexpressing cells resulted in a 3- to 13-fold increase in the expression of the alpha and beta subunit protein of FTase and a 1.5- to 3-fold increase in the level of the enzyme activity compared with untransfected NIH3T3 cells or vector-transfected cells. Further investigations using metabolic labeling indicated that farnesylation of Ras was enhanced in FTase-overexpressing cells. Insulin-like growth factor-I, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) more potently enhanced DNA synthesis and anchorage-dependent growth in FTase-overexpressing cells than in control cells, in a dose-dependent manner. In particular, PDGF and bFGF also induced dose-dependently enhanced colony formation in soft agar in FTase-overexpressing cells. Furthermore, in FTase-transfectants, bFGF stimulated high activation of mitogen-activated protein kinase. Interestingly, FTase-transfectants developed progressive tumors in nude mice. Light and electron microscopy showed that the tumors were characteristic of fibrosarcoma, which were distinct from v-ras-induced tumors. Overexpression of FTase in NIH3T3 cells thus amplifies growth-factor-mediated cell growth and transformation, and FTase-overexpressing cells form tumors in nude mice.     

Effects of KW-3902, a selective and potent adenosine A1 receptor antagonist, on renal hemodynamics and urine formation in anesthetized dogs

Smyth line (SL) chickens develop a spontaneous, autoimmune, posthatch loss of pigment cells (vitiligo) in regenerating feather tissue. Smyth line vitiligo (SLV) is associated with lymphocyte infiltrations prior to and throughout the development of the disorder. It was the purpose of this study to determine the type, relative amounts, and proportions of pulp-infiltrating lymphocytes at various times throughout the growth of regenerating feathers. Feathers were plucked from 8-week-old chickens with and without SLV. Feather pulp cell suspensions were prepared when the regenerating feathers were 2, 3, 4, and 6 weeks of age. Cells were fluorescently labeled using a panel of mouse monoclonal antibodies specific for chicken lymphocytes. Both T and B cells infiltrated the feather pulp of chickens with SLV. T cell levels remained elevated throughout the 6 weeks of feather growth, while B cell levels steadily declined to control levels over the same time. The pulp-infiltrating cells were primarily T cells with an alphabeta T cell receptor expressing the Vbeta1 gene (TCR2+). The ratio between CD4+ and CD8+ cells was 1.42 and 0.75 in 2- and 6-week-old regenerating feathers from chickens with autoimmune SLV, respectively. In non-vitiliginous chickens this ratio was always near 1. These data suggest that TCR2+ T cells play an important role in SLV. CD4+ cells may play a recruiting/activating role, whereas CD8+ cells may have cytotoxic activity specifically directed against melanocytes. Additionally, this is the first report demonstrating the infiltration of B cells into the feather pulp of vitiliginous chickens. These B cells may directly/indirectly contribute to melanocyte destruction in SLV.     

Growth factor effects on apoptosis of rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric epithelium that show increased density during chronic atrophic gastritis. The current study determined cell number and apoptosis of isolated rat ECL cells in response to several growth factors. Isolated ECL cells from fundic mucosa (enrichment >90%) were grown in serum-free medium over 2-5 days. Cell number was determined by mitochondrial formazan production; apoptosis was measured by Tdt-mediated dUTP nick end labeling reaction and DNA fragmentation-based enzyme-linked immunosorbent assay. Immunocytochemistry and RT-PCR demonstrated the presence of epidermal growth factor receptor, neuronal growth factor receptor (type 1), and fibroblast growth factor (FGF) receptor (type 1). Gastrin (EC50, approximately 2 pM), transforming growth factor-alpha (TGF alpha; 10-30 ng/ml), and basic FGF (bFGF; 1-10 ng/ml) increased the total number of cultured ECL cells. bFGF augmented the gastrin (1 pM)-induced response. Beta-neuronal growth factor (10 ng/ml) and bFGF (2 ng/ml) decreased the programed death of ECL cells. Interleukin-1beta (100 pg/ml, 24 h) stimulated apoptosis 2- to 3-fold in ECL cells, and simultaneous incubation with TGF alpha (20 ng/ml) or bFGF (2 ng/ml) significantly inhibited this effect. ECL cells express specific receptors for gastrin, epidermal growth factor, neuronal growth factor, and FGF. bFGF prolonged ECL cell survival by inhibiting spontaneous apoptosis. Our data further indicate that TGF alpha and bFGF increase ECL cell number by inhibiting cytokine-induced programed cell death.