ABSTRACT
BACKGROUND: It remains unknown to what extent consensus molecular subtype (CMS) groups and immune-stromal infiltration patterns improve our ability to predict outcomes over tumor-node-metastasis (TNM) staging and microsatellite instability (MSI) status in early-stage colorectal cancer (CRC). PATIENTS AND METHODS: We carried out a comprehensive retrospective biomarker analysis of prognostic markers in adjuvant chemotherapy-untreated (N = 1656) and treated (N = 980), stage II (N = 1799) and III (N = 837) CRCs. We defined CMS scores and estimated CD8+ cytotoxic lymphocytes (CytoLym) and cancer-associated fibroblasts (CAF) infiltration scores from bulk tumor tissue transcriptomes (CMSclassifier and MCPcounter R packages); constructed a stratified multivariable Cox model for disease-free survival (DFS); and calculated the relative proportion of explained variation by each marker (clinicopathological [ClinPath], genomics [Gen: MSI, BRAF and KRAS mutations], CMS scores [CMS] and microenvironment cells [MicroCells: CytoLym+CAF]). RESULTS: In multivariable models, only ClinPath and MicroCells remained significant prognostic factors, with both CytoLym and CAF infiltration scores improving survival prediction beyond other markers. The explained variation for DFS models of ClinPath, MicroCells, Gen markers and CMS4 scores was 77%, 14%, 5.3% and 3.7%, respectively, in stage II; and 55.9%, 35.1%, 4.1% and 0.9%, respectively, in stage III. Patients whose tumors were CytoLym high/CAF low had better DFS than other strata [HR=0.71 (0.6-0.9); P = 0.004]. Microsatellite stable tumors had the strongest signal for improved outcomes with CytoLym high scores (interaction P = 0.04) and the poor prognosis linked to high CAF scores was limited to stage III disease (interaction P = 0.04). CONCLUSIONS: Our results confirm that tumor microenvironment infiltration patterns represent potent determinants of the risk for distant dissemination in early-stage CRC. Multivariable models suggest that the prognostic value of MSI and CMS groups is largely explained by CytoLym and CAF infiltration patterns.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/genetics , Colorectal Neoplasms/mortality , Microsatellite Instability , Mutation , Transcriptome/drug effects , Tumor Microenvironment/drug effects , Adult , Aged , Aged, 80 and over , Chemotherapy, Adjuvant , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Genomics , Humans , Male , Middle Aged , Neoplasm Staging , Prognosis , Retrospective Studies , Survival Rate , Tumor Microenvironment/genetics , Young AdultABSTRACT
BACKGROUND: Observational studies suggest that higher levels of 25-hydroxyvitamin D3 (25(OH)D) are associated with a reduced risk of colorectal cancer and improved survival of colorectal cancer patients. However, the influence of vitamin D status on cancer recurrence and survival of patients with stage III colon cancer is unknown. PATIENTS AND METHODS: We prospectively examined the influence of post-diagnosis predicted plasma 25(OH)D on outcome among 1016 patients with stage III colon cancer who were enrolled in a National Cancer Institute-sponsored adjuvant therapy trial (CALGB 89803). Predicted 25(OH)D scores were computed using validated regression models. We examined the influence of predicted 25(OH)D scores on cancer recurrence and mortality (disease-free survival; DFS) using Cox proportional hazards. RESULTS: Patients in the highest quintile of predicted 25(OH)D score had an adjusted hazard ratio (HR) for colon cancer recurrence or mortality (DFS) of 0.62 (95% confidence interval [CI], 0.44-0.86), compared with those in the lowest quintile (Ptrend = 0.005). Higher predicted 25(OH)D score was also associated with a significant improvement in recurrence-free survival and overall survival (Ptrend = 0.01 and 0.0004, respectively). The benefit associated with higher predicted 25(OH)D score appeared consistent across predictors of cancer outcome and strata of molecular tumor characteristics, including microsatellite instability and KRAS, BRAF, PIK3CA, and TP53 mutation status. CONCLUSION: Higher predicted 25(OH)D levels after a diagnosis of stage III colon cancer may be associated with decreased recurrence and improved survival. Clinical trials assessing the benefit of vitamin D supplementation in the adjuvant setting are warranted. CLINICALTRIALS.GOV IDENTIFIER: NCT00003835.
Subject(s)
Colonic Neoplasms/pathology , Neoplasm Recurrence, Local , Vitamin D/blood , Adult , Aged , Aged, 80 and over , Colonic Neoplasms/blood , Colonic Neoplasms/mortality , Disease-Free Survival , Female , Humans , Male , Middle Aged , Prospective StudiesABSTRACT
In southern New England, salt marshes are exceptionally vulnerable to the impacts of accelerated sea level rise. Regional rates of sea level rise have been as much as 50% greater than the global average over past decades: a more than four-fold increase over late-Holocene background values. In addition, coastal development blocks many potential marsh migration routes, and compensatory mechanisms relying on positive feedbacks between inundation and sediment deposition are insufficient to counter inundation increases in extreme low turbidity tidal waters. Accordingly, multiple lines of evidence suggest marsh submergence is occurring in southern New England. A combination of monitoring data, field re-surveys, radiometric dating, and analysis of peat composition have established that, beginning in the early and mid-twentieth century, the dominant low marsh plant, Spartina alterniflora, has encroached upwards in tidal marshes, and typical high marsh plants, including Juncus gerardii and Spartina patens have declined, providing strong evidence that vegetation changes are being driven, at least in part, by higher water levels. Additionally, aerial and satellite imagery show shoreline retreat, widening and headward extension of channels, and new and expanded interior depressions. Papers in this special section highlight changes in marsh-building processes, patterns of vegetation loss, and shifts in species composition. The final papers turn to strategies for minimizing and coping with marsh loss by managing adaptively and planning for landward marsh migration. It is hoped that this collection offers lessons that will be of use to researchers and managers on coasts where relative sea level is not yet rising as fast as in southern New England.
ABSTRACT
Northeastern US salt marshes face multiple co-stressors, including accelerating rates of relative sea level rise (RSLR), elevated nutrient inputs, and low sediment supplies. In order to evaluate how marsh surface elevations respond to such factors, we used surface elevation tables (SETs) and surface elevation pins to measure changes in marsh surface elevation in two eastern Long Island Sound salt marshes, Barn Island and Mamacoke Marsh. We compare marsh elevation change at these two systems with recent rates of RSLR and find evidence of differences between the two sites; Barn Island is maintaining its historic rate of elevation gain (2.3± 0.24 mm yr-1 from 2003 to 2013) and is no longer keeping pace with RSLR, while Mamacoke shows evidence of a recent increase in rates (4.2 ± 0.52 mm yr-1 from 1994 to 2014) to maintain its elevation relative to sea level. In addition to data on short-term elevation responses at these marshes, both sites have unusually long and detailed data on historic vegetation species composition extending back more than half a century. Over this study period, vegetation patterns track elevation change relative to sea levels, with the Barn Island plant community shifting towards those plants that are found at lower elevations and the Mamacoke vegetation patterns showing little change in plant composition. We hypothesize that the apparent contrasting trend in marsh elevation at the sites is due to differences in sediment availability, salinity, and elevation capital. Together these two systems provide critical insight into the relationships between marsh elevation, high marsh plant community, and changing hydroperiods. Our results highlight that not all marshes in southern New England may be responding to accelerated rates of RSLR in the same manner.
ABSTRACT
The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.
Subject(s)
Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Binding, Competitive/drug effects , Carrier Proteins/metabolism , Drug Resistance, Neoplasm/drug effects , Indoles/pharmacology , Phosphoproteins/metabolism , Purines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adaptor Proteins, Signal Transducing , Animals , Cell Cycle Proteins , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Colonic Neoplasms/pathology , Eukaryotic Initiation Factors , Female , Humans , MAP Kinase Signaling System/drug effects , Mice , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Ribosomal Protein S6/metabolism , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor Assays , ras Proteins/geneticsABSTRACT
Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Monomeric GTP-Binding Proteins/metabolism , Neuropeptides/metabolism , Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin-Dependent Kinase 2/analysis , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/analysis , Female , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Nude , Multiprotein Complexes , Ras Homolog Enriched in Brain Protein , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/analysis , Up-RegulationABSTRACT
Modified adenoviruses represent a new approach to treatment of gastrointestinal cancer. However, their uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Thus, lack of CAR expression is a potential cause of intrinsic resistance of tumor cells to this type of treatment. To evaluate this, we studied the localization of CAR protein in normal and malignant gastrointestinal tissues. In normal tissues, CAR was concentrated at sites of cell-cell interaction, in particular at the apico-lateral cellular surface. Expression was particularly strong around bile and pancreatic ducts, which is in agreement with CAR's physiological function as a tight-junction protein. In GI malignancies (esophageal, pancreatic, colorectal and liver cancer), expression of the receptor varied substantially. Loss of CAR expression at cell-cell junction was evident in many samples. A significant correlation between CAR expression and histological grade was found, with moderately to poorly differentiated tumors most frequently demonstrating loss or reduction of CAR expression. These data indicate that CAR expression is frequently altered in gastrointestinal malignancy, potentially reducing the efficacy of adenovirus-based therapies.
Subject(s)
Adenoviridae , Enterovirus , Gastrointestinal Neoplasms/metabolism , Receptors, Virus/metabolism , Cell Communication , Cell Differentiation/physiology , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Gastrointestinal Neoplasms/pathology , Humans , Intercellular Junctions/metabolism , Neoplasms/metabolism , Neoplasms/pathologyABSTRACT
Vascular endothelial cell growth factor (VEGF) plays a crucial role in the development of the cardiovascular system and in promoting angiogenesis associated with physiological and pathological processes. Although a great deal is known of the cytoplasmic signaling pathways activated by VEGF, much less is known of the mechanisms through which VEGF communicates with the nucleus and alters the activity of transcription factors. Binding of VEGF to the KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. p38 MAPK/MSK-1 and protein kinase C/p90RSK pathways mediate CREB phosphorylation. Confocal microscopy shows that VEGF-induced phosphorylation of nuclear CREB is blocked by pharmacological inhibition of protein kinase C and p38 mitogen-activated protein kinase signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the nucleus where CREB becomes activated. These results suggest that CREB may play a role in alterations of gene expression important to angiogenesis.
Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins , Endothelial Growth Factors/pharmacology , Lymphokines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Activating Transcription Factor 1 , Cell Line , DNA/metabolism , Electrophoresis, Polyacrylamide Gel , Endothelial Growth Factors/metabolism , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , Lymphokines/metabolism , Microscopy, Confocal , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Receptors, Vascular Endothelial Growth Factor , Serine/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , p38 Mitogen-Activated Protein KinasesABSTRACT
Liver metastases nearly always represent disseminated cancer, and systemic therapies are usually indicated. However, in a minority of patients--some with colorectal cancer, others with selected tumors--management of the hepatic disease may be clinically important and even curative. This review identifies unique patient subgroups and novel treatment approaches that may be indicated in patients with liver metastases.
Subject(s)
Chemoembolization, Therapeutic , Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/therapeutic use , Chemotherapy, Adjuvant , Floxuridine/administration & dosage , Floxuridine/therapeutic use , Fluorouracil/administration & dosage , Fluorouracil/therapeutic use , Hepatic Artery , Humans , Infusions, Intra-Arterial , Liver Neoplasms/drug therapy , Liver Neoplasms/surgeryABSTRACT
Colorectal cancer metastatic to the liver represents an uncommon situation in surgical oncology in which metastasectomy can effect longevity and may lead to cure. Liver resection can be done safely and has drastically improved 5-year survival rates to upwards of 35%, but only a minority of patients is eligible. Advances in imaging techniques facilitate detection of metastases and improve patient selection. For unresectable patients, and as an adjunct to resection, local disease control may be achieved with cryosurgery, radiofrequency ablation, or regional chemotherapy delivered by way of infusion through the hepatic artery. Areas of ongoing investigation include regional gene therapy and interference with tumor growth by inhibition of angiogenesis.
Subject(s)
Adenocarcinoma/secondary , Adenocarcinoma/therapy , Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Liver Neoplasms/therapy , Adenocarcinoma/diagnosis , Adenocarcinoma/mortality , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/mortality , Colorectal Neoplasms/surgery , Combined Modality Therapy , Female , Hepatectomy/methods , Humans , Liver Neoplasms/diagnosis , Liver Neoplasms/mortality , Male , Neoplasm Staging , Prognosis , Survival RateSubject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/secondary , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Floxuridine/administration & dosage , Liver Neoplasms/drug therapy , Liver Neoplasms/secondary , Colorectal Neoplasms/pathology , Dexamethasone/administration & dosage , Humans , Infusion Pumps, Implantable , Infusions, Intra-ArterialABSTRACT
Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.
Subject(s)
Endothelial Growth Factors/pharmacology , Gene Expression Regulation/drug effects , Lymphokines/pharmacology , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Cells, Cultured , Culture Media, Conditioned/pharmacology , Down-Regulation/drug effects , Enzyme Activation/drug effects , Humans , Iodine Radioisotopes , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/drug effects , Protein Binding , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth FactorsABSTRACT
PURPOSE: Management of anal high-grade squamous intraepithelial lesions is controversial. Anal and cervical high-grade squamous intraepithelial lesions are similar in that they occur in transitional squamous epithelium, are associated with human papilloma virus infection, and have increased incidence in the immunocompromised population. Ablation of cervical high-grade squamous intraepithelial lesions is preferred, but similar ablation or excision of anal high-grade squamous intraepithelial lesions may compromise bowel control; thus, there is a need to define the malignant potential of anal high-grade squamous intraepithelial lesions. METHODS: We analyzed 50 paraffin sections of normal anoderm, anal low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and anal squamous-cell carcinoma. Microvessels were detected immunohistochemically with von Willebrand factor and counted manually along the epithelial-stromal junction. Proliferation and apoptosis were determined in the epithelial cells with MIB-1 antibody immunostaining and the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, respectively. RESULTS: Microvascular density was significantly greater in anal high-grade squamous intraepithelial lesions (mean, 0.50 vessels/cm) vs. normal anoderm (mean, 0.21 vessels/cm; P = 0.0017, Mann-Whitney U test). The proliferative percentages were greater in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous-cell carcinoma (mean, 20.4, 21.8, and 23.6 percent) vs. normal anoderm (mean, 14.4 percent), although not significantly (P = 0.06, Kruskal-Wallis statistic). Although the mean proliferative proportions were similar in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, the apoptotic proportion was lower for high-grade squamous intraepithelial lesions than low-grade squamous intraepithelial lesions (10.13 vs. 19.96 percent, respectively; P = NS, Mann-Whitney U test). CONCLUSIONS: Angiogenesis, increased proliferation, and decreased apoptosis occur in anal high-grade squamous intraepithelial lesions as they do in the cervix before the development of malignancy. These biologic markers support the importance of anal high-grade squamous intraepithelial lesions as a potential premalignant lesion warranting surgical intervention.
Subject(s)
Anus Neoplasms/blood supply , Apoptosis/physiology , Carcinoma in Situ/blood supply , Carcinoma, Squamous Cell/blood supply , Cell Division/physiology , Neovascularization, Pathologic/pathology , Anal Canal/blood supply , Anal Canal/pathology , Anus Neoplasms/pathology , Biopsy , Carcinoma in Situ/pathology , Carcinoma, Squamous Cell/pathology , Humans , Microcirculation/pathology , Neoplasm StagingABSTRACT
A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Endothelial Growth Factors/metabolism , Lymphokines/metabolism , Membrane Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Amino Acid Sequence , Carrier Proteins/genetics , Cells, Cultured , Cloning, Molecular , Endothelium, Vascular/metabolism , Humans , Isoenzymes/metabolism , Membrane Proteins/genetics , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Protein Binding , RNA, Messenger/metabolism , Receptors, Vascular Endothelial Growth Factor , Sequence Alignment , Signal Transduction , Type C Phospholipases/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , src Homology DomainsABSTRACT
This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.
Subject(s)
Cell Division/physiology , Endothelium, Vascular/cytology , Mitogens/physiology , Protein Serine-Threonine Kinases , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Cell Adhesion Molecules/metabolism , Cells, Cultured , Endothelial Growth Factors/physiology , Endothelium, Vascular/enzymology , Endothelium, Vascular/metabolism , Enzyme Activation , Epidermal Growth Factor/physiology , Fibroblast Growth Factors/physiology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Isoenzymes/metabolism , Lymphokines/physiology , Neovascularization, Physiologic , Phospholipase C gamma , Protein Kinase C/metabolism , Protein Kinase C-epsilon , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptors, Vascular Endothelial Growth Factor , Recombinant Proteins/metabolism , Type C Phospholipases/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.
Subject(s)
RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Base Sequence , DNA Primers/genetics , Gene Expression , Humans , Liver/metabolism , Male , Paraffin Embedding , Prostate/metabolism , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Time Factors , Transcription, GeneticABSTRACT
Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.
Subject(s)
Endothelial Growth Factors/antagonists & inhibitors , Endothelium, Vascular/drug effects , Lymphokines/antagonists & inhibitors , Protein Tyrosine Phosphatases/physiology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Growth Factor/antagonists & inhibitors , Tumor Necrosis Factor-alpha/pharmacology , Cell Division/drug effects , Cells, Cultured , DNA/biosynthesis , Endothelium, Vascular/cytology , Humans , Intracellular Signaling Peptides and Proteins , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Receptor Protein-Tyrosine Kinases/analysis , Receptor Protein-Tyrosine Kinases/drug effects , Receptors, Growth Factor/analysis , Receptors, Growth Factor/drug effects , Receptors, Vascular Endothelial Growth Factor , Signal Transduction/drug effects , Tyrosine/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.
Subject(s)
Coenzyme A Ligases/metabolism , Mitochondria, Liver/enzymology , Animals , Carboxylic Acids/metabolism , Cattle , Chromatography, Gel , Chromatography, Ion Exchange , Coenzyme A Ligases/chemistry , Fatty Acids/metabolism , Humans , Hydrogen-Ion Concentration , Kinetics , Substrate Specificity , Xenobiotics/metabolismABSTRACT
Comprehensive information about the molecular cytogenetic changes in metastases of colorectal cancer is not yet available. To define such changes in metastases, we measured relative DNA sequence copy numbers by comparative genomic hybridization (CGH). Samples from 27 liver metastases and 6 synchronous primary tumors were analyzed. An average of 9.9 aberrations per tumor was found in the metastases. Gains of chromosome arms 20q (85%), 13q (48%), 7p (44%), and 8q (44%) and losses of chromosome arms 18q (89%), 8p (59%), 1p (56%), and 18p (48%) were detected most frequently. Chromosomes 14 and 15 were lost in 26% and 30% of the metastases, respectively. No consistent differences were observed between primary tumors and synchronous metastases. Fluorescence in situ hybridization (FISH) was used for further characterization of gains of chromosome arm 20q. Touch preparations of 13 tumors that had demonstrated 20q gain with CGH were examined with FISH by use of a set of probes mapping to different parts of 20q. A probe for 20p was used as a reference. FISH showed relative gain of at least one 20q locus in 12 of the tumors. High-level gains were detected in 38% of the tumors, preferentially for probes mapping to band 20q13. Our CGH data indicate that colorectal metastases show chromosomal changes similar to those that have been reported for primary tumors. Chromosomal losses were seen at higher frequency, particularly for chromosomes 14 and 15. By FISH, we identified subregions on chromosome arm 20q that are frequently involved in DNA amplifications in colorectal cancer and that may harbor candidate proto-oncogenes.
Subject(s)
Chromosome Aberrations/genetics , Chromosomes, Human/genetics , Colorectal Neoplasms/genetics , In Situ Hybridization, Fluorescence/methods , Liver Neoplasms/secondary , Nucleic Acid Hybridization/methods , Adult , Aged , Aged, 80 and over , Chromosome Disorders , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 15/genetics , Chromosomes, Human, Pair 18/genetics , Chromosomes, Human, Pair 20/genetics , Chromosomes, Human, Pair 7/genetics , Chromosomes, Human, Pair 8/genetics , Colorectal Neoplasms/pathology , Female , Humans , Liver Neoplasms/genetics , Male , Middle Aged , Neoplasm StagingABSTRACT
BACKGROUND: Volume-outcome relations have been established for several complex therapies. However, few studies have examined volume-outcome relations for high-risk procedures in general surgery, such as hepatectomy for hepatocellular carcinoma (HCC). OBJECTIVE: To evaluate the relation between hospital volume and outcome for patients undergoing hepatectomy for HCC. DESIGN: Retrospective cohort study. SETTING: All acute-care hospitals in California. PATIENTS: Hospital discharge data were analyzed for each patient in California who underwent major hepatic resection for HCC from January 1, 1990, through December 31, 1994. Hospitals were grouped according to number of hepatectomies performed at each center during the 5-year study. MAIN OUTCOME MEASURES: Outcome measures included operative mortality and length of hospital stay. Regression analyses were used to adjust for differences in patient mix. RESULTS: Five hundred seven patients underwent hepatectomy for HCC during the study. Hepatic resections were performed in 138 hospitals, with an overall in-hospital mortality rate of 14.8%. Three quarters of patients were treated at hospitals that average 3 or fewer hepatic resections for HCC per year. These low-volume providers represent 97.1% of all hospitals treating patients with HCC statewide. Significant reductions in risk-adjusted operative mortality rates (22.7%-9.4%; P = .002, multiple logistic regression) and risk-adjusted length of stay (14.3-11.3 days; P = .03, multiple linear regression) were observed as hospital volume increased. CONCLUSIONS: Low operative mortality and length of stay were associated with high-volume centers. These data support regionalization of high-risk procedures in general surgery, such as hepatectomy for HCC.