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Renal cell carcinoma (RCC) is one of the few human cancers whose incidence is increasing. The disease regularly progresses asymptomatically and is frequently metastatic upon presentation, thereby necessitating the development of an early method of detection. A metabolomic approach for biomarker detection using urine as a biofluid is appropriate since the tumor is located in close proximity to the urinary space. By comparing the composition of urine from individuals with RCC to control individuals, differences in metabolite composition of this biofluid can be identified, and these data can be utilized to create a clinically applicable and, possibly, bedside assay. Recent studies have shown that sample handling and processing greatly influences the variability seen in the urinary metabolome of both cancer and control patients. Once a standard method of collection is developed, identifying metabolic derangements associated with RCC will also lead to the investigation of novel targets for therapeutic intervention. The objective of this review is to discuss existing methods for sample collection, processing, data analysis, and recent findings in this emerging field.

Kidney cancer is frequently metastatic on presentation at which point the disease is associated with a 95% mortality. Assessment of tumor grade on pathological examination is the most powerful means for prognostication as well as for stratification of patients into those who might respond to conventional or targeted therapy. Although there exist several grading systems in common use, all suffer from significant disparity among observers. In an attempt to objectify this process as well as to acquire grade-specific mechanistic information, we performed LC-MS/MS-based proteomics analysis on 50 clear cell kidney cancers equally distributed among normal tissues and Fuhrman grades 1–4. Initial experiments confirmed the utility of using archived formalin-fixed paraffin-embedded samples for LC-MS/MS-based proteomics analysis, and the LC-MS/MS findings were validated by extensive immunoblotting. We now show that changes among many biochemical processes and pathways are strongly grade-dependent with the glycolytic and amino acid synthetic pathways highly represented. In addition, proteins relating to acute phase and xenobiotic metabolism signaling are highly represented. Self-organized mapping of proteins with similar patterns of expression led to the creation of a heat map that will be useful in grade characterization as well as in future research relating to oncogenic mechanisms and targeted therapies for kidney cancer.

Renal cell carcinoma (RCC) accounts for 11,000 deaths per year in the United States. When detected early, generally serendipitously by imaging conducted for other reasons, long term survival is generally excellent. When detected with symptoms, prognosis is poor. Under these circumstances, a screening biomarker has the potential for substantial public health benefit. The purpose of this study was to evaluate the utility of urine metabolomics analysis for metabolomic profiling, identification of biomarkers, and ultimately for devising a urine screening test for RCC. Fifty urine samples were obtained from RCC and control patients from two institutions, and in a separate study, urine samples were taken from 13 normal individuals. Hydrophilic interaction chromatography-mass spectrometry was performed to identify small molecule metabolites present in each sample. Cluster analysis, principal components analysis, linear discriminant analysis, differential analysis, and variance component analysis were used to analyze the data. Previous work is extended to confirm the effectiveness of urine metabolomics analysis using a larger and more diverse patient cohort. It is now shown that the utility of this technique is dependent on the site of urine collection and that there exist substantial sources of variation of the urinary metabolomic profile, although group variation is sufficient to yield viable biomarkers. Surprisingly there is a small degree of variation in the urinary metabolomic profile in normal patients due to time since the last meal, and there is little difference in the urinary metabolomic profile in a cohort of pre- and postnephrectomy (partial or radical) renal cell carcinoma patients, suggesting that metabolic changes associated with RCC persist after removal of the primary tumor. After further investigations relating to the discovery and identity of individual biomarkers and attenuation of residual sources of variation, our work shows that urine metabolomics analysis has potential to lead to a diagnostic assay for RCC.

The diagnosis of cancer by examination of the urine has the potential to improve patient outcomes by means of earlier detection. Due to the fact that the urine contains metabolic signatures of many biochemical pathways, this biofluid is ideally suited for metabolomic analysis, especially involving diseases of the kidney and urinary system. In this pilot study, we test three independent analytical techniques for suitability for detection of renal cell carcinoma (RCC) in urine of affected patients. Hydrophilic interaction chromatography (HILIC-LC-MS), reversed-phase ultra performance liquid chromatography (RP-UPLC-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) all were used as complementary separation techniques. The combination of these techniques is best suited to cover a very large part of the urine metabolome by enabling the detection of both lipophilic and hydrophilic metabolites present therein. In this study, it is demonstrated that sample pretreatment with urease dramatically alters the metabolome composition apart from removal of urea. Two new freely available peak alignment methods, MZmine and XCMS, are used for peak detection and retention time alignment. The results are analyzed by a feature selection algorithm with subsequent univariate analysis of variance (ANOVA) and a multivariate partial least squares (PLS) approach. From more than 2000 mass spectral features detected in the urine, we identify several significant components that lead to discrimination between RCC patients and controls despite the relatively small sample size. A feature selection process condensed the significant features to less than 30 components in each of the data sets. In future work, these potential biomarkers will be further validated with a larger patient cohort. Such investigation will likely lead to clinically applicable assays for earlier diagnosis of RCC, as well as other malignancies, and thereby improved patient prognosis.

Under some conditions, p21(Waf1/Cip1) plays an assembly factor role for the cyclins and cyclin-dependent kinases, and recent reports demonstrate that p21 can act as an anti-apoptotic protein. Thus, it is logical to exploit this function of p21 as an anti-cancer target. We have performed a pilot study showing that daily subcutaneous injection of a phosphorothioate antisense p21 oligodeoxynucleotide, which we have previously shown to attenuate p21 levels in vitro, into nude mice who have been implanted with highly metastatic breast cancer cells results in inhibition of tumor growth and angiogenesis. Inhibition of in vitro endothelial capillary formation confirms that these oligodeoxynucleotides have a direct effect upon tumor angiogenesis. The attractiveness of our novel approach to breast cancer therapy, which capitalizes on the anti-apoptotic function of p21, derives from the ease of transfection of antisense oligodeoxynucleotides as well as the observations that p21(-/-) mice do not develop spontaneous tumors, making techniques exploiting the assembly factor and anti-apoptotic role of p21 worthy of further study against breast cancer.

The cyclin-dependent kinase inhibitors (CKI) interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but we and others have recently shown that these molecules can exert permissive effects on cell cycle transit as well. The p53 protein induces transcription of the p21(Waf1/Cip1) gene, but whether p53 has any effect on the stimulatory versus inhibitory state of p21(Waf1/Cip1) toward cell growth is not known. The focus of the current study was to examine the effect of p21(Waf1/Cip1) inhibition on growth in cells which possess an inactive p53 protein. We found that there was significant and specific inhibition of p21(Waf1/Cip1) protein transcription in human squamous carcinoma A431 cells after transfection of an antisense p21(Waf1/Cip1) oligodeoxynucleotide, yet there was no significant growth inhibition in these cells after stimulation with 10% serum or with PDGF-BB, in contrast to what was observed in vascular smooth muscle (VSM) cells. Furthermore, there was no attenuation of either cyclinD/cdk4 association or of Rb hyperphosphorylation after antisense p21(Waf1/Cip1) oligodeoxynucleotide transfection, suggesting that an alternate pathway exists to allow association and phosphorylation of these cell cycle components in the absence (or with lower levels) of p21(Waf1/Cip1). Thus, the permissive effect of p21(Waf1/Cip1) toward growth is dependent on cell type, and active p53 is likely required for this effect.

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.

The kidney is responsible for regulation of water and electrolyte balance, filtration and absorption of plasma proteins, and control of blood volume and pressure. Homeostasis achieved by the kidney is controlled in large part by the action of hormones or proteins on specific transmembrane receptors. Conversely, many renal diseases, including that resulting from atherosclerosis, are characterised by scarring and abnormal proliferation of cellular components of the kidney, and these processes are mediated in large part by these same receptors. The G protein–coupled receptors constitute a large and diverse class of proteins, characterised by the possession of seven transmembrane-spanning domains. These receptors bind polypeptide growth factors, which function to transmit a variety of signals from the extracellular to the intracellular milieu. The receptor-associated G proteins utilised by the kidney derive their specificity not only by activating or inhibiting various second-messenger molecules, but also by their location on particular cell types. In this review, several G protein–coupled receptors will be discussed from the perspective of their importance to kidney function and to the pathogenesis of renal disease, atherosclerosis, and hypertension.

Mitogenesis induced by most polypeptide growth factors is mediated by either G-protein- or tyrosine kinase-linked receptor pathways. Thrombin, a potent mitogen for vascular smooth muscle cells, activates a G-protein-coupled receptor but also requires tyrosine kinase activity for its mitogenic effect (Weiss, R. H., and Nuccitelli, R. (1992) J. Biol. Chem. 267, 5608-5613). Since this growth factor elicits a synergistic effect on DNA synthesis when applied to cells concurrently with basic fibroblast growth factor (bFGF), we suspected that the two growth factors have points of convergence in their signaling pathways. We now show that when 1 unit/ml thrombin is removed after an incubation period of from 4 h to 15 min prior to 15 ng/ml bFGF addition, its synergistic effect with bFGF on mitogenesis in vascular smooth muscle cells is preserved. Furthermore, appearance of bFGF in the cellular lysate is maximal 1 h after the addition of 1 unit/ml thrombin. While monoclonal antibody to bFGF inhibits thrombin's mitogenic effect by 63% at 30 micrograms/ml, it lacks an inhibitory effect on platelet-derived growth factor-BB-induced mitogenesis. The inhibitory effect of bFGF antibody on thrombin's growth is completely reversed by the addition of bFGF. These data demonstrate that the presence of bFGF is essential for thrombin to exert its full mitogenic effect in vascular smooth muscle cells, providing an example of a system where a tyrosine kinase-linked growth factor receptor system can act as an essential intermediary in the mitogenic signaling pathway of a G-protein-coupled receptor.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.

In neonatal vascular smooth muscle (VSM) cells, activation of protein kinase C can block the mitogenic response to α-thrombin. The molecular mechanism for this growth inhibition was investigated by looking at early transcriptional events in the cell cycle. Both thrombin and phorbol-12-myristate-13-acetate (PMA) induced mRNA for the c-myc oncogene; peak levels of expression were found 4–5 h after exposure to either agent. When thrombin and PMA were added together, c-myc expression was increased synergistically; down-regulation of protein kinase C suppressed induction of c-myc by thrombin. Thus, c-myc expression varied inversely with cell growth under these conditions. Thrombin and PMA also both induced expression of mRNA for the PDGF-A chain over 4–7h. As for c-myc, PMA and thrombin synergistically increased expression of the PDGF A-chain under conditions where PMA inhibits thrombin-induced DNA synthesis. Thus, mitogenesis and early growth-related gene expresion was dissociated during PMA-mediated growth inhibition.