Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephritogenic, carcinogenic, and teratogenic action. Epidemiological studies indicate that OTA may be involved in the pathogenesis of different forms of human nephropathies. Previously we have shown that OTA activates extracellular signal-regulated kinases 1 and 2, members of the mitogen-activated protein kinases (MAPK) family, in the C7-clone but not in the C11-clone of renal epithelial Madin-Darby canine kidney (MDCK) cells. Here we show that nanomolar concentrations of OTA lead to activation of a second member of the MAPK family, namely, c-jun amino-terminal-kinase (JNK) in MDCK-C7 cells but virtually not in MDCK-C11 cells, as determined by kinase assay and Western blot. Furthermore, OTA potentiated the effect of tumor necrosis factor-alpha on JNK activation. In parallel to its effects on JNK, nanomolar OTA induced apoptosis in MDCK-C7 cells but not in MDCK-C11 cells, as determined by DNA fragmentation, DNA ladder formation, and caspase activation. In addition, OTA potentiated the proapoptotic action of tumor necrosis factor-alpha. Our data provide additional evidence that OTA interacts in a cell type-specific way with distinct members of the MAPK family at concentrations where no acute toxic effect can be observed. Induction of apoptosis via the JNK pathway can explain some of the OTA-induced changes in renal function as well as part of its teratogenic action.

Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice.

Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED(50) = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED(50) = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.

Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase.

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

Constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 induces epithelial dedifferentiation and growth inhibition in madin-darby canine kidney-C7 cells.

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.

Ochratoxin A-induced stimulation of extracellular signal-regulated kinases 1/2 is associated with Madin-Darby canine kidney-C7 cell dedifferentiation.

The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species of Aspergillus and Penicillium. OTA has the ability to disturb Madin-Darby canine kidney (MDCK) cell pH homeostasis, leading to intracellular alkalinization and morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress. Because alkali-induced epithelial dedifferentiation of MDCK-C7 cells is associated with an increase in the activity of extracellular signal-regulated kinases (ERK), we performed experiments that investigated a possible role for ERK1 and ERK2 as intracellular signaling molecules mediating some of the mycotoxin's effects on renal epithelia. We studied the effects of OTA on ERK1/2 phosphorylation and activation, as well as on cell morphology by using cloned MDCK-C7 and MDCK-C11 cells. In MDCK-C7 cells, but not in MDCK-C11 cells, OTA led to a time-dependent and concentration-dependent increase in ERK1/2 phosphorylation. OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells occurred at concentrations of 500 nM, started after 2 hr and was maximal after 8 hr. Furthermore, after 8 hr of incubation, 500 nM and 1 microM OTA significantly increased ERK1/2 activity in MDCK-C7 but not in MDCK-C11 cells. This OTA-stimulated ERK1/2 phosphorylation and ERK1/2 activation in MDCK-C7 cells was partially inhibited by the synthetic mitogen-activated protein kinase kinase (MKK or MEK) inhibitor PD098059. Transepithelial resistance and lactate dehydrogenase release remained unaltered after incubation in the presence of 1 microM OTA for 8 hr or of 100 nM OTA for 24 hr, so it is unlikely that these OTA effects on ERK1/2 are due to secondary toxic effects of the mycotoxin. Interestingly, OTA-induced long-term activation of ERK1/2 in MDCK-C7 cells was associated with epithelial dedifferentiation, as assessed by analysis of vectorial solute and water transport as well as cell morphology. In contrast, MDCK-C11 cells, which do not show significant increases in ERK1/2 phosphorylation and ERK1/2 activity in response to OTA, retained their epithelial phenotype under identical experimental conditions. Taken together, our data demonstrate an epithelial dedifferentiation of MDCK-C7 cells, but not of MDCK-C11 cells, after long-term incubation in the presence of OTA, a result associated with the ability of this mycotoxin to stimulate ERK1/2 in MDCK-C7 cells but not in MDCK-C11 cells. We conclude that OTA-induced activation of ERK1/2 could be an important intracellular signaling pathway that mediates some of the mycotoxin's effects on renal epithelia.

Loss of cytokeratin expression and formation of actin stress fibers in dedifferentiated MDCK-C7 cell lines.

MDCK-C7 cells dedifferentiated either by transient alkaline stress (C7F cells) or by transfection with a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (C7caMEK1 cells) were analyzed by western blot and immunofluorescence microscopy to compare the expression of different cytokeratins, vimentin, and alpha-smooth muscle actin. Expression of all cytokeratins tested, the type II-neutral and basic cytokeratins CK5, CK7, CK8 as well as the type I-acidic keratins CK17 and CK19, was substantially reduced in dedifferentiated cell lines C7F and C7caMEK1 when compared with epithelial wild-type MDCK-C7 cells or mock-transfected MDCK-C7 cells. While vimentin expression was detected in all of the four MDCK-C7 cell lines examined, only the dedifferentiated cell lines C7F and C7caMEK1, which have been reported to express highly active ERK2, exhibited formation of alpha-smooth muscle actin-containing stress fibers. Taken together our results show that, associated with an increase in ERK2 activity, an epithelial to mesenchymal dedifferentiation occurred in both MDCK-C7F cells and caMEK1-transfected MDCK-C7 cells.

Topical capsaicin for treatment of hemodialysis-related pruritus.

Pruritus is a significant problem for many patients undergoing long-term hemodialysis. Topical capsaicin depletes and prevents the reaccumulation of substance P in peripheral sensory neurons. Substance P functions in the transmission of pain and probably itch sensations. An open-label, uncontrolled trial and a double-blind, vehicle-controlled trial were conducted to evaluate the efficacy and safety of capsaicin 0.025% cream in the treatment of localized areas of pruritus in patients undergoing long-term hemodialysis. Eight of nine evaluable patients in the open-label trial reported marked relief or complete resolution of itching during the study period, and two of five evaluable patients in the double-blind trial reported complete resolution of itching in the capsaicin-treated arm with no or minimal improvement in the vehicle-treated arm. Twelve patients in the open-label trial and two in the double-blind trial were unevaluable. No serious treatment-related adverse reactions occurred.

N-(5-fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxamide. A novel, systemically active antitumor agent effective against 3LL Lewis lung carcinoma.

N-(5-Fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxam ide (1) is a member of a series of amides found to substantially increase lifespan in mice bearing established micrometastatic 3LL Lewis lung carcinoma. Amide 1 is effective after either oral or intraperitoneal dosing in acute, subacute, or chronic regimens. 1 is well tolerated in this model with an excellent therapeutic index relative to the cytotoxic anticancer drug adriamycin.

Use of young nude mice for selection of subpopulations of cells with increased metastatic potential from nonsyngeneic neoplasms.

The purpose of these studies was to investigate whether young (3-wk-old) nude mice, which lack functional T-lymphocytes and demonstrate low natural killer cell activity, could serve as in vivo models for the selection of metastatic subpopulations of cells from heterogeneous allogeneic melanomas. Three-week-old BALB/cAnN or N:NIH(S) nude mice received iv injections of single cells harvested from either the B16 or K-1735 melanoma. Individual pulmonary metastases were harvested 4 weeks later and implanted sc into nude mice to expand the population. Cells from each of these metastases colonized in the lungs of 3-week-old nude mice and 6-week-old normal syngeneic mice with significantly higher efficiency than did cells from the parent tumors. It was concluded that, in addition to being a useful in vivo model for ascertaining the metastatic potential of neoplasms, the healthy young nude mouse could be used for selecting and maintaining tumor cell variants of high metastatic potential room heterogeneous allogeneic tumors.

Active specific immunotherapy of established micrometastases with BCG plus tumor cell vaccines: effective treatment of BCG side effects with isoniazid.

Intradermal vaccinations with Bacillus Calmette-Guérin (BCG) and viable but nontumorigenic syngeneic hepatocarcinoma cell vaccines were used successfully to treat micrometastatic disease experimentally induced in inbred guinea pigs at 100 times the minimal lethal dose. Several complications have been associated with the use of viable BCG organisms in the treatment of cancer patients and, in this animal model, intradermal administration of BCG uniformly results in severe ulceration and eschar formation at the injection sites leading to secondary microbial invasion and regional lymphadenopathy. We now report the use of isoniazid (Nydrazid) as part of an active specific immunization regimen. Incorporation of isoniazid into the immunization procedure for two weeks alleviates or reduces the side effects of BCG infection. Moreover, with proper consideration of BCG dosage, isoniazid does not impair the efficacy of the BCG plus tumor cell vaccines.

Separation of guinea pig peripheral blood lymphocytes by discontinuous density gradient centrifugation using Ficoll-metrizoate.

We present a simple method for the recovery of lymphocytes from guinea pig peripheral blood by discontinuous density gradient centrifugation using Ficoll-metrizoate solutions. This technique is formulated specifically for the routine preparation of guinea pig lymphocytes for in vitro cultivation and is capable of recovering 40-60% of available lymphocytes. Whole, heparinized (10-100 U/ml) blood was drawn from strain 2 guinea pigs by cardiac puncture, diluted 1 : 4 with Ca2,Mga free Hanks' balanced salt solution (CMF-HBSS) containing EDTA (5 mM) and gentamicin (50 microgram/ml), and incubated at room temperature for 30-60 min to promote leukocyte disaggregation. Five volumes of diluted blood were layered onto 2 vol of Ficoll-metrizoate adjusted to a density of 1.107 g/ml with sodium metrizoate solution. A band of mononuclear cells (80-90% lymphocytes, 10-20% monocytes, and less than 2% granulocytes) formed at the interface after centrifugation (400 X g, 20-40 min, room temperature). More than 95% of the cells were viable by trypan blue exclusion. Lymphocytes recovered from as little as 3-5 ml whole blood were more sensitive to antigen- or mitogen-activated transformation than leukocyte suspensions obtained by dextran-citrate sedimentation with or without nylon column filtration.

Production of human chorionic gonadotropin and its subunits by human tumors growing in nude mice.

The production and secretion of human chorionic gonadotropin (HCG) and its subunits by human tumors growing in nude mice have been examined. JAR choriocarcinoma cells growing in nude mice produce both free alpha subunit and complete HCG, but there is a decrease in the amount of free alpha subunit relative to complete HCG produced in vivo compared to HCG subunit production by these cells growing in culture. Cell lines that produce only free alpha subunit in culture (HeLa cervical carcinoma, ChaGo bronchogenic carcinoma, and BT-20 breast carcinoma) continue to produce primarily free alpha subunit in vivo, but a small amount of HCG-beta/HCG is detectable in the 24-hr urine collected from mice bearing HeLa or ChaGo tumors. CBT cells derived from a glioblastoma multiforme produce both alpha and HCG-beta/HCG in vivo. This represents a distinct shift from the pattern of HCG subunit production by CBT cells in culture because cultured CBT cells produce only free beta subunit and do not synthesize either free alpha or complete HCG. Thus, for human tumors growing in nude mice, there appears to be a shift toward more complete HCG production and a decrease in free subunit production as compared to the pattern observed for cultured cells.

Studies of the paw test and tumor-associated immunosuppression in mice.

Specific paw test reactivity to tumor cells was transferred to normal mice using lymphoid cells or serum from mice with a growing or surgically excised methylcholanthrene-induced transplantable sarcoma. When the donors had been sensitized to PPD and to tumor, normal recipients of lymphoid cells became reactive by specific paw test to tumor cells and PPD. Normal recipients of serum from the same donors also became reactive to tumor cells. Normal recipients of serum from tumor-bearing mice developed depressed PHA paw tests. Adoptive transfer with cells or serum of paw test reactivity to tumor and PPD was unsuccessful when recipients had large tumors. This was the case when the recipients had either tumor of the same line or that of a separate line that did not cross-react antigenically.

Thromboembolic disease in renal allograft recipients. What is its clinical significance?

Detailed analysis of the clinical data and autopsy material of 100 consecutive renal transplant recipients revealed significant thromboembolic disease in 25 patients and a total of 41 complications. In six of them, thromboembolism was associated with sepsis. Nine patients died (20% of total number of deaths) due to a primary thromboembolic event. The incidence of pulmonary embolism was 14%; myocardial infarction, 3%; cerebrovascular disease, 4%; renal artery thrombosis, 2%; renal vein thrombosis, 3%; thrombophlebitis/deep vein thrombosis, 13%; and miscellaneous, 2%. The incidence of thromboembolism was higher in patients older than 40 years of age (P = .02) and during the earlier months after transplantation. We summarize the general incidence and mortality related to thromboembolism and discuss the factors predisposing the graft recipient to thromboembolic disease. Prevention and therapy of this complication should decrease the morbidity and mortality in graft recipients and enhance the success of renal transplantation.

Growth responses of Escherichia coli to the surfactant dodecyl benzene sulfonate.

When strains of Escherichia coli are grown in broth cultures containing the anionic surfactant sodium dodecyl benzene sulfonate (NaDDBS), they exhibit unique growth responses. After 20 to 24 hr of incubation, they become slimy and viscous, and an addition of ethanol to the supernatant liquid yields a distinctive white, fibrous precipitate. The production of this material was shown to be dependent on the presence of NaDDBS in the medium. This precipitate from E. coli ATCC 11303 was found to contain 41 to 53% protein, 10 to 11% deoxyribonucleic acid, 6.8 to 7.4% ribonucleic acid, 15 to 25% carbohydrate, and 9% lipid. It is distinctive from naturally occurring E. coli slimes in several respects. Our data suggest that its formation is the primary result of the leakage of intracellular components into the medium. However, the rate of cell proliferation indicates a partial but not complete or lethal lysis. A limited utilization of NaDDBS as a carbon source was also shown.