Cardiovascular risk and kidney function profiling using conventional and novel biomarkers in young adults: the African-PREDICT study.

BACKGROUND: Low- and middle-income countries experience an increasing burden of chronic kidney disease. Cardiovascular risk factors, including advancing age, may contribute to this phenomenon. We (i) profiled cardiovascular risk factors and different biomarkers of subclinical kidney function and (ii) investigated the relationship between these variables. METHODS: We cross-sectionally analysed 956 apparently healthy adults between 20 and 30 years of age. Cardiovascular risk factors such as high adiposity, blood pressure, glucose levels, adverse lipid profiles and lifestyle factors were measured. Various biomarkers were used to assess subclinical kidney function, including estimated glomerular filtration rate (eGFR), urinary albumin, uromodulin and the CKD273 urinary proteomics classifier. These biomarkers were used to divide the total population into quartiles to compare extremes (25th percentiles) on the normal kidney function continuum. The lower 25th percentiles of eGFR and uromodulin and the upper 25th percentiles of urinary albumin and the CKD273 classifier represented the more unfavourable kidney function groups. RESULTS: In the lower 25th percentiles of eGFR and uromodulin and the upper 25th percentile of the CKD273 classifier, more adverse cardiovascular profiles were observed. In multi-variable adjusted regression analyses performed in the total group, eGFR associated negatively with HDL-C (ß= -0.44; p < 0.001) and GGT (ß= -0.24; p < 0.001), while the CKD273 classifier associated positively with age and these same risk factors (age: ß = 0.10; p = 0.021, HDL-C: ß = 0.23; p < 0.001, GGT: ß = 0.14; p = 0.002). CONCLUSION: Age, lifestyle and health measures impact kidney health even in the third decade.

Understanding glomerular diseases through proteomics.

INTRODUCTION: Chronic kidney disease is avery common and complex chronic disease. Uncovering the pathological patterns of CKD on the molecular level of bio-fluids and tissue appears to be both vital and promising for a more favorable outcome. We reviewed recently discovered proteomics biomarkers for CKD to provide new insight into disease pathology. AREAS COVERED: We review the application of proteome analysis in the context of CKD with various etiologies within the last 5 years. Proteins and peptides associated with CKD as derived from multiple sources (urine, blood and tissue) are reported along with their various biological pathways. EXPERT OPINION: A systematic and theoretical comprehension of the CKD pathology is essential for its successful management. The underlying complexity of the disease further requires specific conditions for reliable and interpretable results. In this context, clinical proteomics has resulted in first encouraging findings in CKD. A more complete understanding of the biological pathways related to the disease, based on the scope of a holistic proteomic approach, could improve substantially the management of CKD, especially when in conjunction with the current trend of personalized medicine.

Characteristics of high- and low-risk individuals in the PRIORITY study: urinary proteomics and mineralocorticoid receptor antagonism for prevention of diabetic nephropathy in Type 2 diabetes.

AIM: To compare clinical baseline data in individuals with Type 2 diabetes and normoalbuminuria, who are at high or low risk of diabetic kidney disease based on the urinary proteomics classifier CKD273. METHODS: We conducted a prospective, randomized, double-blind, placebo-controlled international multicentre clinical trial and observational study in participants with Type 2 diabetes and normoalbuminuria, stratified into high- or low-risk groups based on CKD273 score. Clinical baseline data for the whole cohort and stratified by risk groups are reported. The associations between CKD273 and traditional risk factors for diabetic kidney disease were evaluated using univariate and logistic regression analysis. RESULTS: A total of 1777 participants from 15 centres were included, with 12.3% of these having a high-risk proteomic pattern. Participants in the high-risk group (n=218), were more likely to be men, were older, had longer diabetes duration, a lower estimated GFR and a higher urinary albumin:creatinine ratio than those in the low-risk group (n=1559, P<0.02). Numerical differences were small and univariate regression analyses showed weak associations (R2 < 0.04) of CKD273 with each baseline variable. In a logistic regression model including clinical variables known to be associated with diabetic kidney disease, estimated GFR, gender, log urinary albumin:creatinine ratio and use of renin-angiotensin system-blocking agents remained significant determinants of the CKD273 high-risk group: area under the curve 0.72 (95% CI 0.68-0.75; P<0.01). CONCLUSIONS: In this population of individuals with Type 2 diabetes and normoalbuminuria, traditional diabetic kidney disease risk factors differed slightly between participants at high risk and those at low risk of diabetic kidney disease, based on CKD273. These data suggest that CKD273 may provide additional prognostic information over and above the variables routinely available in the clinic. Testing the added value will be subject to our ongoing study. (European Union Clinical Trials Register: EudraCT 2012-000452-34 and Clinicaltrials.gov: NCT02040441).

Urine peptidomic biomarkers for diagnosis of patients with systematic lupus erythematosus.

Background Systematic lupus erythematosus (SLE) is characterized with various complications which can cause serious organ damage in the human body. Despite the significant improvements in disease management of SLE patients, the non-invasive diagnosis is entirely missing. In this study, we used urinary peptidomic biomarkers for early diagnosis of disease onset to improve patient risk stratification, vital for effective drug treatment. Methods Urine samples from patients with SLE, lupus nephritis (LN) and healthy controls (HCs) were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS) for state-of-the-art biomarker discovery. Results A biomarker panel made up of 65 urinary peptides was developed that accurately discriminated SLE without renal involvement from HC patients. The performance of the SLE-specific panel was validated in a multicentric independent cohort consisting of patients without SLE but with different renal disease and LN. This resulted in an area under the receiver operating characteristic (ROC) curve (AUC) of 0.80 ( p < 0.0001, 95% confidence interval (CI) 0.65-0.90) corresponding to a sensitivity and a specificity of 83% and 73%, respectively. Based on the end terminal amino acid sequences of the biomarker peptides, an in silico methodology was used to identify the proteases that were up or down-regulated. This identified matrix metalloproteinases (MMPs) as being mainly responsible for the peptides fragmentation. Conclusions A laboratory-based urine test was successfully established for early diagnosis of SLE patients. Our approach determined the activity of several proteases and provided novel molecular information that could potentially influence treatment efficacy.

A urinary peptide biomarker set predicts worsening of albuminuria in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Microalbuminuria is considered the first clinical sign of kidney dysfunction and is associated with a poor renal and cardiovascular prognosis in type 2 diabetes. Detection of patients who are prone to develop micro- or macroalbuminuria may represent an effective strategy to start or optimise therapeutic intervention. Here we assessed the value of a urinary proteomic-based risk score (classifier) in predicting the development and progression of microalbuminuria. METHODS: We conducted a prospective case-control study. Cases (n = 44) and controls (n = 44) were selected from the PREVEND (Prevention of Renal and Vascular End-stage Disease) study and from the Steno Diabetes Center (Gentofte, Denmark). Cases were defined by transition from normo- to microalbuminuria or from micro- to macroalbuminuria over a follow-up of 3 years. Controls with no transitions in albuminuria were pair-matched for age, sex and albuminuria status. A model for the progression of albuminuria was built using a proteomic classifier based on 273 urinary peptides. RESULTS: The proteomic classifier was independently associated with transition to micro- or macroalbuminuria (OR 1.35 [95% CI 1.02, 1.79], p = 0.035). The classifier predicted the development and progression of albuminuria on top of albuminuria and estimated GFR (eGFR, area under the receiver operating characteristic [ROC] curve increase of 0.03, p = 0.002; integrated discrimination index [IDI]: 0.105, p = 0.002). Fragments of collagen and α-2-HS-glycoprotein showed significantly different expression between cases and controls. CONCLUSIONS/INTERPRETATION: Although limited by the relatively small sample size, these results suggest that analysis of a urinary biomarker set enables early renal risk assessment in patients with diabetes. Further work is required to confirm the role of urinary proteomics in the prevention of renal failure in diabetes.

Proteome analysis in hematology using capillary electrophoresis coupled on-line to mass spectrometry.

Proteome analysis, the key technology for biomarker discovery, continues to gain importance in clinical diagnosis and follow-up. In this review we describe proteome analysis in the context of allogeneic, hematopoietic stem cell transplantation concentrating on capillary electrophoresis coupled on-line to mass spectrometry.

Proteomics in gerontology: current applications and future aspects--a mini-review.

BACKGROUND: Aging is closely related to the onset of chronic diseases, such as coronary artery disease, diabetic nephropathy or different types of malignancies, reflecting the demand for novel biomarkers to manage theses diseases. OBJECTIVE: The analysis of the human proteome for biomarkers has made considerable advances in the last years. METHODS: We describe the main technological approaches taken, their advantages and disadvantages. RESULTS: We will review the different clinical sources of material and attempt to highlight the different challenges and approaches associated with these. Age-related changes in the proteome have been described and were found to be highly similar to changes associated with chronic diseases. We will give several examples on the successful application of proteomics in the diagnosis, prognosis and therapy of these chronic diseases. CONCLUSIONS: A boost in disease-related proteomic information is expected in the very near future, and will also result in its broad clinical application. However, this view appears to be dependent on the strict adherence to proper technological/analytical parameters, correct statistics, and large databases that allow comparison of datasets provided by different scientists. Clearly, the proteome is by far too complex to be tackled by one laboratory on its own.

[Proteome analysis of undiluted vitreous humor in patients with branch retinal vein occlusion]. / Proteomanalyse unverdünnter Glaskörperflüssigkeit bei Patienten mit einem Venenastverschluss.

BACKGROUND: The pathophysiological mechanisms of macular edema secondary to branch retinal vein occlusion (BRVO) remain unclear. OBJECTIVES: To analyze the protein profile of human vitreous of patients with BRVO and to identify specific dysregulated proteins. MATERIALS AND METHODS: Undiluted vitreous humor samples from patients with treatment naïve BRVO and 15 controls with idiopathic floaters were analyzed in this clinical-experimental study using capillary electrophoresis coupled to a mass spectrometer (CE-MS) and tandem mass spectrometry (MS/MS). Quantitative analysis of the dysregulated proteins was performed with enzyme-linked immunosorbent assay (ELISA). Protein-protein interactions were depicted with the STRING database. RESULTS: A total of 84 proteins were found in the human vitreous samples of 15 patients with BRVO and 15 controls. In all, 14 proteins were significant when comparing the signal intensities of BRVO and control samples. Six significant dysregulated proteins with p < 0.001 were further verified with ELISA. Clusterin, complement factor C3, prostaglandin-H2 D­isomerase and vitronectin were significantly upregulated in the BRVO group and opticin was downregulated. The protein interactions analysis showed associations with inflammatory cascades, matrix changes, mechanisms of cell survival und death. CONCLUSIONS: The results of the study reveal that the proteomic composition of vitreous humor differed significantly between the patients with BRVO and the controls. Whether the identified proteins may serve as potential biomarkers for pathophysiology, diagnostics or therapy should be examine in further studies.

Preferential activation of microsomal diacylglycerol/protein kinase C signaling during glucose treatment (De Novo phospholipid synthesis) of rat adipocytes.

Glucose has been reported to increase the de novo synthesis of diacylglycerol (DAG) and translocate and activate protein kinase C (PKC) in rat adipocytes. Presently, we examined the major subcellular site of PKC translocation/activation in response to glucose-induced DAG. Glucose rapidly increased DAG content and PKC enzyme activity in microsomes, but not in plasma membranes or other membranes, during a 30-min treatment of rat adipocytes. This glucose-induced increase in microsomal DAG was attended by increases in immunoreactive PKC alpha, beta, and epsilon. Glucose-induced activation of DAG/PKC signaling in microsomes was not associated with a change in the translocation of Glut-4 transporters from microsomes to the plasma membrane, a biological response that is known to be stimulated by agonists, e.g., phorbol esters, which increase DAG/PKC signaling in plasma membranes, as well as in microsomes. In conclusion, an increase in de novo phospholipid synthesis, as occurs during glucose treatment of rat adipocytes, primarily activates DAG/PKC signaling in microsomes; moreover, this signaling response and biological consequences thereof may differ from those of agonists that primarily stimulate DAG/PKC signaling in the plasma membrane.

Addition of constitutive c-myc expression to Abelson murine leukemia virus changes the phenotype of the cells transformed by the virus from pre-B-cell lymphomas to plasmacytomas.

Abelson murine leukemia virus (A-MuLV), a retrovirus that expresses the v-abl oncogene, characteristically induces pre-B-cell lymphomas following in vivo infection of BALB/c mice or in vitro infection of suspensions of fetal liver or bone marrow cells. ABL-MYC, a retrovirus that expresses both v-abl and c-myc, induces solely plasmacytomas in BALB/c mice. To investigate how the addition of overexpression of c-myc to that of v-abl accomplishes this dramatic change in the phenotype of the cells transformed by these closely related retroviruses, we utilized helper-free A-MuLV (psi 2) and ABL-MYC (psi 2) in vitro to infect suspensions of cells from different lymphoid tissues and purified immature and purified mature B cells. As expected, A-MuLV(psi 2) induced only pre-B-cell lymphomas in vivo and in vitro when immature B cells were present. ABL-MYC(psi 2), on the other hand, produced only plasmacytomas, even when purified immature B lymphocytes were infected in vitro. Although the A-MuLV(psi 2)-induced pre-B-cell lymphomas express easily detectable levels of c-myc mRNA, maturation into more-mature forms of B lymphocytes is blocked. The constitutively overexpressed c-myc in the ABL-MYC retrovirus abrogates this block, permits maturation of infected immature B cells, and yields transformed plasma cells.

Negative regulation of Raf-1 by phosphorylation of serine 621.

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein.

We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

Mechanism of inhibition of Raf-1 by protein kinase A.

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.

Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity.

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-epsilon (PKC epsilon). We recently have shown that Raf-1 is constitutively activated in PKC epsilon transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKC epsilon induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKC epsilon activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKC epsilon and Raf-1. PKC epsilon, but not PKC alpha, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/epsilon cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/epsilon cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKC epsilon. PKC epsilon did not induce translocation of Raf-1 to the membrane. Furthermore, PKC epsilon did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFbeta, a growth factors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.

The catalytic domain of PKC-epsilon, in reciprocal PKC-delta and -epsilon chimeras, is responsible for conferring tumorgenicity to NIH3T3 cells, whereas both regulatory and catalytic domains of PKC-epsilon contribute to in vitro transformation.

Protein kinase C-epsilon (PKC-epsilon) has been shown to increase growth and cause malignant transformation when overexpressed in NIH3T3 cells, whereas PKC-delta reduced fibroblast growth. Two reciprocal chimeric proteins (PKC-epsilondelta and PKC-deltaepsilon were constructed by exchanging the regulatory and catalytic domains of PKC-delta and -epsilon and were stably overexpressed in NIH3T3 cells. Fibroblasts that overexpressed either chimera showed maximum cell density and morphology that were intermediate between cells overexpressing PKC-delta and those that overexpressed PKC-epsilon. Moreover, all lines that expressed chimeras were capable of anchorage-independent growth in the presence of TPA, which indicated that both the regulatory and catalytic domains of PKC-epsilon could independently induce NIH3T3 transformation, although the combination of both domains, as found in PKC-epsilon, was the most active form. In contrast, the translocation pattern and ability to induce tumors in nude mice was attributable to the catalytic domains exclusively. In particular, cells that expressed PKC-deltaepsilon retained PKC-epsilon's full potency of tumorgenicity when injected into nude mice. In sum, our findings not only reinforce the concept that only certain PKC isozymes contribute to carcinogenesis but also show that different domains of PKCs mediate the physiologically distinguishable events of transformation and tumorgenesis.

Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells.

We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.

Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.

A raf/myc virus immortalized macrophage cell line which supports the growth of B-cell and B-cell hybridomas.

Using a combination of raf and myc oncogenes co-expressed by the recombinant retrovirus J-2 we have generated and characterized a cell line which very efficiently supports the growth of B-cells and B-cell hybridomas. Murine spleen cells were cultured under in vitro immunization conditions favoring the short term proliferation of splenic B lymphocytes and infected with J-2 virus. Screening of immortalized spleen cell pools for the capability to support long term B cell growth in vitro led to the selection of a clonal cell line termed alpha ChyJ2. The presence of macrophage specific features and surface markers suggest that alpha ChyJ2 belongs to the macrophage lineage. alpha ChyJ2 cells constitutively produce low levels of IL-1 like activity and high levels of IL-6. Expression of specific mRNAs as well as production of IL-1 alpha, IL-1 beta and IL-6 are inducible with LPS. Expression or production of other cytokines including IL-2, IL-3, IL-4, IL-5, TGF beta and GM-CSF could not be detected. As the biological effects of alpha ChyJ2 supernatant cannot be fully explained by the described pattern of cytokine production, participation of other, yet uncharacterized, factors is possible. Using alpha ChyJ2 as feeder cells for in vitro as well as in vivo immunizations increased the number of antibody secreting B-cell clones 2 to 15 fold, respectively.