Effect of herbimycin A on growth and pp60c-src activity in human colon tumor cell lines.

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125ng ml-1 dose of the antibiotic caused greater than 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dose-dependent inactivation of pp60c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60c-src kinase activity preceded changes in pp60c-src steady-state levels. Growth and pp60c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60c-src tyrosine kinase activity may be important in growth control of colon tumor cells.     

Inhibition of formation of microtubular paracrystals in HeLa-S3 cells by neocarzinostatin.

The effect of an antitumor antibiotic, neocarzinostatin (NCS), on the formation of microtubular paracrystals (PC) induced by vinblastine sulfate, 10 mug/ml, in HeLa-S3 cells was examined by phase-contrast microscopy. The pretreatment of HeLa-S3 cells with NCS, 5 to 50 mug/ml, for 4 hr prevented the PC formation, and there was a dose response of NCS to the degree of inhibition. When the same inhibitory effect on PC formation was examined with other antitumor agents at high doses (50 mug/ml), colchicine was found to be one of the most effective agents, like NCS. Puromycin, antimycin, adriamycin, cytochalasin B, and cycloheximide revealed moderate activity, and the other antibiotics, such as mitomycin C, bleomycin, and rifampicin, did not show any effect at all. NCS was a unique antibiotic that inhibited PC formation among inhibitors of DNA synthesis. It was suggested that NCS affects the microfibrillar-microtubular proteins system in vivo, resulting in the inhibitions of organization of spindle fibers from microtubules at the G2 phase in HeLa cells.     

New Insight into Oncoprotein-Targeted Antitumor Effect: Herbimycin a as an Antagonist of Protein Tyrosine Kinase against Ph1-Positive Leukemia Cells

Herbimycin A, a benzoquinonoid anasamycin antibiotic, has been shown to reserve the oncogenic phenotypes of p60^v-src transformed cells by the virtue of the inhibition of src protein tyrosine kinase. Furthermore, we previously demonstrated that herbimycin A displayed the antitumor activity on Ph¹-positive leukemia cells and bcr/abl oncoprotein-associated transformed murine hematopoietic cells with the transfection of a retroviral vector expressing bcr/abl. Herbimycin A showed preferential inhibition on the in vitro growth of Ph¹-positive leukemia cells and bcr/abl oncoprotein-associated murine hematopoietic cells through the inhibition of bcr/abl tyrosine kinase activity and the reduction of subsequent phosphotyrosyl proteins. Recently, from the view of investigating the oncogenic significance or of developing a future clinical application in malignancies, several developing agents targeted against oncoprotein have been tried. We reviewed the present progress in the mechanism of oncoprotein-targeted antitumor effects and focused on herbimycin A-induced antitumor activity on Ph¹-positive leukemia cells.     

Transformation of NIH3T3 cells by Rous sarcoma virus occurs with high efficiency in the absence of proviral rearrangements or amplification.

NIH3T3 cells could be transformed by a mammaltropic strain of Rous sarcoma virus (RSV) with an efficiency 10(3) times greater than that observed in Balb/c 3T3 cells or other mammalian cell lines and almost identical to that of chick embryo fibroblasts. In infected NIH3T3 cells a single, properly integrated, provirus was sufficient to induce focus formation; moreover, kinase activity of pp60v-src and tyrosine phosphorylation of cellular proteins could be detected very soon after infection in the majority of cells. On the other hand, in transformed foci from RSV-infected Balb/c 3T3 cells both rearrangements and amplification of proviral sequences were frequently detected. Accordingly, expression of pp60v-src and ensuing tyrosine phosphorylation of cellular proteins occurred, at high levels, only in a minority of the infected cells. Furthermore, by using a murine retrovirus carrying the v-src oncogene and an independent selectable marker, we found that Balb/c 3T3 cells were transformed with a 100-fold lower efficiency than NIH3T3 cells, yet the majority of infected untransformed Balb/c 3T3 cells expressed active pp60v-src. These findings are consistent with the existence in most mammalian cell lines of a major restriction to v-src-induced transformation, operating at the level of proviral expression, that is apparently absent in NIH3T3 cells.     

Transformation by polyoma virus middle T antigen

The transforming protein of polyoma virus, middle T antigen, is a membrane-associated phosphoprotein. Middle T forms a complex with, and is phosphorylated by, a cellular tyrosine kinase pp60c-src. Mutant analysis suggests that formation of this complex is critical to transformation by polyoma. Middle T binding causes pp60c-src to autophosphorylate at novel sites in its amino terminus, and increases the specific activity of the enzyme. However, at least one non-transforming mutant of middle T, dl1015, can also activate pp60c-src in these ways. This suggests that properties of middle T other than the ability to activate pp60c-src are also necessary for transformation. These properties may include the ability to associate with a phosphatidylinositol kinase, and/or with a protein of 61 kDa. The mechanism by which middle T activates pp60c-src may involve its ability to alter the phosphorylation state of the enzyme, and thus interfere with the regulation of pp60c-src activity in vivo. Polyoma virus transformed cells might then be a good model system for investigating the control of pp60c-src activity as well as defining substrates of the enzyme.     

Genistein and quercetin increase connexin43 and suppress growth of breast cancer cells

Connexin proteins form gap junctions, which permit direct exchange of cytoplasmic contents between neighboring cells. Evidence indicates that gap junctional intercellular communication (GJIC) is important for maintaining homeostasis and preventing cell transformation. Furthermore, connexins may have independent functions including tumor growth suppression. Most tumors express less connexins, have reduced GJIC and have increased growth rates compared with non-tumorigenic cells. The purpose of this study was to determine whether common flavonoids, genistein and quercetin, increase connexin43 (Cx43) levels, improve GJIC and suppress growth of a metastatic human breast tumor cell line (MDA-MB-231). Quercetin (2.5, 5 microg/ml) and genistein (0.5, 2.5, 15 microg/ml) upregulated Cx43 but failed to increase GJIC. Cx43 localized to the plasma membrane following genistein treatment (2.5, 15 mug/ml). In contrast, Cx43 aggregated in the perinuclear region following quercetin treatment (0.5, 2.5, 5, 15 microg/ml). Both genistein (15 microg/ml) and quercetin (2.5, 5, 15 microg/ml) significantly reduced MDA-MB-231 cell proliferation. In summary, genistein and quercetin increase Cx43 and suppress MDA-MB-231 cell proliferation at physiologically relevant concentrations. These results demonstrate that genistein and quercetin are potential anti-breast cancer agents.     

The amino-terminal region of pp60c-src has a modulatory role and contains multiple sites of tyrosine phosphorylation.

The c-src gene product from either platelet-derived growth factor treated cells or from polyomavirus-infected cells migrates anomalously on gels, displays enhanced kinase activity, and contains additional sites of tyrosine phosphorylation within its amino-terminus. To probe the importance of these post-translational modifications, each of the five amino-terminal tyrosine residues (residues 90, 92, 131, 136, and 149) was altered to phenylalanine using site-directed mutagenesis. Except for the 136F variant, the mutants displayed enhanced kinase activity, albeit at a low level shown insufficient to induce focus formation in NIH3T3 cells. Mutagenesis of residues adjacent to tyrosines 90 and 92 also resulted in variants with enhanced kinase activity indicating that the region and not the tyrosines per se, may be involved in regulating this activity. Fingerprinting analysis demonstrated that the enhanced kinase activity brought about by these mutations occurred through a mechanism which appears to be independent of the phosphorylation state of Tyr 416 and possibly Tyr 527. Upon treatment of cells with vanadate both amino-terminal variants and transforming mutants of pp60c-src displayed a slower migrating form, designated p60+. Hence, the appearance of these p60+ proteins may be elicited either by mutations that enhance the transforming activity of pp60c-src or by perturbations within the amino-terminal region of the enzyme. The retarded mobility of p60+ was shown to be due in part to additional tyrosine phosphorylations residing at its amino-terminus. The demonstration that p60+ could be resolved into multiple bands and that amino-terminal fragments containing phosphorylated tyrosine residues were obtained regardless of which of the five tyrosines in the region was altered to phenylalanine indicates that there are multiple sites of tyrosine phosphorylation within the amino-terminal region of pp60c-src.     

Immunofluorescent localization and quantitation of 3'-azido-2',3'-dideoxythymidine (azt) incorporated into chromosomal DNA of human, hamster and mouse-cell lines

The incorporation of [H-3] AZT into the DNA of cultured hamster (Chinese hamster ovary, CHO) mouse (NIH 3T3) and human (HL60) cells, was studied by radiolabeling, radioimmunoassay, and localization of the drug in chromosomes by immunohistochemistry. Incorporation of AZT into DNA, in cells exposed to 0.8 mM [H-3] AZT for 24 hours, showed values of 0.82 pmol/mug DNA for HL60, 0.84 pmol/mug DNA for CHO and 4.49 pmol/mug for NIH 3T3 cells. Similar results were obtained by radioimmunoassay with an anti-AZT antibody. Indirect immunofluorescence using the same anti-AZT antiserum demonstrated the chromosomal localization of AZT and showed higher concentrations of AZT in the telomeric regions of CHO cell chromosomes.     

Reversal of transformed phenotypes by herbimycin A in src oncogene expressed rat fibroblasts

We studied the effectiveness of herbimycin A, an inhibitor of the function of the src oncogene, to reverse the various transformed phenotypes in normal rat kidney (NRK) cells integrating temperature-sensitive v-src (ts/NRK). Elevated glucose transport in ts/NRK cells at a permissive temperature (33 degrees C) was decreased by herbimycin in 8 h to near the level that was observed either in ts/NRK grown at a nonpermissive temperature (39 degrees C) or in untransformed NRK cells at either temperature. Herbimycin caused no significant decrease in glucose uptake in ts/NRK cells grown at 39 degrees C. The effects of herbimycin on serum- and anchorage-independent growth properties of ts/NRK cells and of NRK cells integrating K-ras (KNRK) were also examined. With ts/NRK cells grown at 33 degrees C, the inhibition of cell growth by herbimycin became more pronounced when the serum concentration in the medium was lowered. With KNRK cells, in contrast, almost the same extent of cell growth inhibition was exerted by herbimycin irrespective of the serum concentration. Furthermore, with ts/NRK cells grown at 33 degrees C, herbimycin inhibited the colony formation in the soft agar medium more strongly than on a solid support. No such differential effects were observed with KNRK cells under similar conditions. These results suggest that herbimycin specifically acts on cells expressing the src oncogene and reverses various transformed characteristics to the normal ones.     

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.     

Antitumor activity of ER-51785, a new peptidomimetic inhibitor of farnesyl transferase: synergistic effect in combination with paclitaxel

Inhibitors of ras farnesylation have been extensively studied in the preclinical stage, and some of them are being developed in the clinic. Herein, we describe the antitumor activity of a new farnesyl transferase inhibitor, ER-51785. In vitro, ER-51785 selectively inhibited farnesyl transferase activity (IC50 = 77 nM) compared with geranylgeranyl transferase I activity (IC50 = 4200 nM). In cells, ER-51785 inhibited posttranslational processing of H-ras with IC50 = 28 nM, but not that of rap 1A at concentrations up to 50 microM. This compound also strongly inhibited colony formation of H-ras-transformed NIH 3T3 fibroblasts and EJ-1 bladder carcinoma cells. In vivo, ER-51785 showed potent tumor regression activity against EJ-1 xenografts but only modest activity against MIA PaCa-2 xenografts. Treatment of ER-51785 in combination with paclitaxel exhibited synergistic effects against colony formation and tumor growth of MIA PaCa-2 cells. The results presented herein support the idea that farnesyl transferase inhibitors alone and in combination with other chemotherapeutic agents have the potential to be developed as therapies for tumors expressing H-ras or K-ras oncogenes.     

Differential inhibition of the epidermal growth factor-, platelet-derived growth factor-, and protein kinase C-mediated signal transduction pathways by the staurosporine derivative CGP 41251.

The microbial alkaloid staurosporine is a potent but nonselective inhibitor of protein kinases. The derivative CGP 41251 has been shown to exert a high degree of selectivity for inhibition of protein kinase C activity. Both compounds are powerful inhibitors of proliferation of both normal and transformed cells in vitro and exert antitumor efficacy in vivo. In this work we have studied the mode of action of these compounds by analyzing their effects on early events in the induction of proliferation by different growth stimuli. Both drugs blocked the phorbol ester-induced expression of the c-fos proto-oncogene. The effect of CGP 41251 was reversible, since its removal led to a normal expression of c-fos mRNA in response to phorbol 12-myristate 13-acetate. Submicromolar concentrations of CGP 41251 and staurosporine directly inhibited both the platelet-derived growth factor (PDGF) receptor autophosphorylation and the c-fos mRNA expression induced by PDGF stimulation of intact BALB/c 3T3 cells. In contrast, ligand-induced epidermal growth factor receptor autokinase activity in A431 carcinoma cells and epidermal growth factor-dependent c-fos mRNA expression were relatively insensitive to inhibition by CGP 41251. Staurosporine suppressed signal generation by the epidermal growth factor receptor by reducing overall levels of the receptor. We conclude that CGP 41251 is a potent reversible inhibitor of protein kinase C and PDGF-mediated signal transduction. It inhibits the kinase activity of both protein kinase C and the PDGF receptor tyrosine kinase and the subsequent signaling cascade. The broad inhibition of kinases by staurosporine is also reflected at the cellular level and might contribute to the high toxicity of this compound, in comparison to CGP 41251.     

Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation

Epidermal growth factor receptor (EGFR) and Src tyrosine kinase cooperate in regulating EGFR-mediated cell signaling and promoting cell transformation and tumorigenesis in pathological conditions. Activation of Src is tightly regulated by the C-terminal Src kinase (Csk). The Csk-binding protein (Cbp) is a ubiquitously expressed transmembrane protein. Its functions include suppression of T-cell receptor activation through recruiting Csk and inhibiting Src family kinase (SFK). However, a potential role of Cbp in EGF-induced cell activities has not been investigated. Here, we report that EGF-stimulation-induced Cbp tyrosine phosphorylation followed by Cbp-Csk association, in a SFK-dependent manner. Expression of wild-type (wt) Cbp remarkably suppressed EGF-induced activation of Src, ERK1/2, and Akt-1 enzymes, and NIH3T3 cell transformation, as well as colony formation of a breast cancer cell line (MDA-MB-468) in soft agar. In contrast, expression of CbpY317F or knockdown endogenous Cbp in NIH3T3 cells by RNA interference significantly enhanced EGF-induced activation of these enzymes and cell transformation. In addition, overexpression of multiple receptor tyrosine kinases (RTKs)-induced Cbp tyrosine phosphorylation. These results demonstrate that Cbp functions as a negative regulator of cell transformation and tumor cell growth through downregulation of Src activation, suggesting that Cbp might be broadly involved in RTKs-activated signaling pathways and tumorigenesis.     

Lovastatin and tumor necrosis factor-alpha exhibit potentiated antitumor effects against Ha-ras-transformed murine tumor via inhibition of tumor-induced angiogenesis

Lovastatin, a drug commonly used in the treatment of hypercholesterolemia, has previously been reported to exert potentiated antitumor activity when combined with either tumor necrosis factor-alpha (TNF-alpha), cisplatin or doxorubicin in a melanoma model in mice. Since lovastatin interferes with the function of ras oncogene-encoded (Ras) proteins, we have investigated the antitumor activity of lovastatin and TNF-alpha using a Ha-ras-transformed murine tumor model. In in vitro studies, lovastatin inhibited the growth of cells transformed with Ha-ras oncogene (Ras-3T3 and HBL100-ras cells) more effectively than control NIH-3T3 and HBL100-neo cells. In in vivo experiments, the Ras-3T3 tumor demonstrated significantly increased sensitivity to combined treatment with both lovastatin (50 mg/kg) and TNF-alpha (1 microg/day) compared with either agent alone. Combined treatment with both agents also resulted in greater inhibition of blood-vessel formation. Ras-3T3 tumor cells produced increased amounts of vascular endothelial growth factor (VEGF) and lovastatin effectively suppressed VEGF production by these cells. Our results suggest that lovastatin increases antitumor activity of TNF-alpha against tumor cells transformed with v-Ha-ras oncogene via inhibition of tumor-induced blood-vessel formation.     

Specific antigrowth effect of interferon on mouse cells transformed by murine sarcoma virus

The growth rate of NIH/3T3 mouse fibroblasts transformed by the Moloney strain of murine sarcoma virus was investigated following interferon (IFN) treatment. These cells were found to be sensitive to the antigrowth effect of IFN as indicated by a slower growth rate in its presence. The effect was most efficiently expressed when cells were grown at low serum concentrations, e.g., 2.5%. Likewise, IFN treatment caused a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. In contrast, these effects were observed only to a very minor extent with uninfected NIH/3T3 cells or with NIH/3T3 cells chronically infected with murine leukemia virus. In addition, IFN treatment significantly decreased the cloning efficiency of murine sarcoma virus-transformed cells in semi-solid agar. Furthermore, an even stronger effect on the cloning efficiency was observed in liquid medium supplied with 2.5% serum, indicating a direct inhibitory effect on the growth of these cells. Under these conditions, NIH/3T3 or NIH/3T3(MLV) cells remained unaffected by IFN treatment, nor was this effect evident when the transformed cells were grown in the presence of 10% serum. A 4-fold increase in the level of (2'-5')oligoadenylate synthetase following IFN treatment was observed in murine sarcoma virus-transformed cells as compared to either NIH/3T3 or NIH/3T3(MLV) cells.     

In vivo treatment of mutant FLT3-transformed murine leukemia with a tyrosine kinase inhibitor.

Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 microM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.     

Cell-killing Activity and Kinetic Analysis of a Novel Antitumor Compound NC-190, a Benzo[a]phenazine Derivative

A novel antitumor compound, N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]-phenazine-6-carboxamide sodium salt (NC-190), was evaluated for antitumor activity in vitro against cultured tumor cell lines, and the kinetics of cell killing was elucidated. NC-190 strongly inhibited the growth of all of 3 murine tumor cell lines, 7 human tumor cell lines and 2 normal cell lines. With continuous exposure, the 50% inhibition concentrations were in the range of 0.005–0.06 μg/ml, except for KATO-III (2.15 μ g/ml). By colony-forming assay, concentrations of NC-190 giving 90% cell kill (IC₉₀) at various exposure times were obtained with HeLa S3 cells. The plot of IC₉₀exposure time on a log-log scale was linear for NC-190 with a slope of -1, which is typical for cell cycle phase-nonspecific agents. A 2 h treatment with NC-190 induced a rapid reduction in cell viability at doses of more than 3 μ g/ml. At the dose where colony formation was completely inhibited, cell viability was persistently reduced to below 20% during the cell culture period. NC-190 cauced a dose- and time-dependent reduction in DNA synthesis. The inhibitions of RNA and protein synthesis were less than that of DNA synthesis. Spectroscopic studies of NC-190 mixed with calf thymus DNA demonstrated that NC-190 was capable of interacting with DNA. However, DNA thermal denaturation studies suggested that intercalation of NC-190 was weak in comparison with those of classical intercalating drugs.     

Transformation-specific Decrease of Phosphorylation of 80K Protein, a Substrate of Protein Kinase C, in NIH3T3 Cells

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two-dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c- raf or H- ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)-stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N- ras , K- ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone-inducible H- ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation.     

Transformation-specific decrease of phosphorylation of 80K protein, a substrate of protein kinase C, in NIH3T3 cells

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two-dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c-raf or H-ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)-stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N-ras, K-ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone-inducible H-ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation.