Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.

Enrichment of mutant KRAS alleles in pancreatic juice by subtractive iterative polymerase chain reaction.

SUMMARY: The detection of mutant tumor genes holds great promise for an early diagnosis of primary tumors and residual malignant disease. When few tumor cells are present with an excess of nonmalignant cells of the same lineage, the excess of wild-type alleles over mutant tumor alleles presents an analytical problem. The subtractive iterative PCR (siPCR) assay presents a new approach to solving this problem. To achieve an enrichment of mutant alleles, wild-type alleles are removed by differential hybridization to complementary oligonucleotides spanning the region of the gene in which point mutations are expected. The nonbound fraction is reamplified by PCR. By iterating this process, mutant alleles can be detected in the presence of an excess of wild-type alleles with high sensitivity. To prove the feasibility of siPCR, pancreatic juice samples were analyzed for KRAS mutations. Pancreatic juice obtained from patients with pancreatic carcinoma or chronic pancreatitis during endoscopic retrograde cholangiopancreatography was analyzed for point mutations in codons 12 and 13 of the KRAS gene. In each of six samples from tumor patients, mutations in codon 12 were detected. One of nine samples from patients with chronic pancreatitis scored positive.

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor.

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.

Enrichment of mutant alleles by chromatographic removal of wild type alleles: a new principle for the detection of alleles with unknown point mutations at excess of wild type alleles.

In human carcinomas, mutations that alter tumour genes such as the KRAS, P53, or APC genes, are mostly point mutations. The detection of mutant alleles of tumour genes in specimens such as urine, pancreatic juice, sputum, and stool holds great promise for an early diagnosis of cancer. In addition, the detection of mutant tumour genes in tissue samples, such as lymph nodes or resection margins, may allow a sensitive diagnosis of residual malignant disease. However, the reliable detection of mutant alleles in excess of wild type alleles remains an unresolved analytical problem when the mutations are not known a priori. In the present communication, a new approach is described which makes possible the detection of unknown point mutations in tumour genes at excess of wild type alleles. The method is based on the removal of wild type alleles by hybridisation to immobilised complementary oligonucleotides. Using this approach, an enrichment of mutant KRAS, P53 and APC alleles of one mutant in up to 10(3) normal alleles has been achieved. Parallel miniaturised separation units with oligonucleotides complementary to defined sequences of a wild type allele should allow the detection of unknown point mutations as well as small insertions or deletions which occur in the sequence range covered by the oligonucleotides.

Dysregulated expression of CD66a (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.

CD66a (BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen family that has been shown to be down-regulated in colorectal, prostate, and breast cancers. The purpose of the present study was to determine its expression pattern in the normal human endometrium and in endometrial neoplasia. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody on a series of 24 normal endometrial samples and 47 endometrial carcinomas. Strong CD66a expression was observed in glandular and luminal epithelial cells of the normal endometrium with a consistent localization at the apical poles of these cells throughout the cycle. In late secretory (premenstrual) phase, loss of cellular polarity resulted in a membranous expression pattern in some glandular cells. In the analyzed tumor samples increasing areas with a complete loss of expression were observed with increasing malignancy grade. The apical expression pattern of the normal epithelium was changed to a membranous all-around pattern in 55% of the tumors, mostly in solid areas. This change correlated with malignancy grade and could be observed in 3 of 15 G1 tumors, 4 of 12 G2 tumors, 11 of 12 G3 tumors, and 8 of 8 serous-papillary carcinomas. Areas with membranous expression pattern could be observed along with areas with a normal apical expression pattern in lower grade carcinomas and with areas with complete loss of expression in high grade tumors. Northern blot analysis showed a loss of mRNA expression in tumor samples and HEC-1B endometrial adenocarcinoma cells. Loss of protein expression in the tumor samples was also observed by Western blot. In conclusion, CD66a protein expression is dysregulated in endometrial carcinomas, showing reduction or loss of expression with increasing malignancy grade and a change from the apical to a membranous localization.

Methods for detection of point mutations: performance and quality assessment. The IFCC Scientific Division, Committee on Molecular Biology Techniques.

We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (j) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.

Dysregulation of carcinoembryonic antigen group members CGM2, CD66a (biliary glycoprotein), and nonspecific cross-reacting antigen in colorectal carcinomas. Comparative analysis by northern blot and in situ hybridization.

Genes coding for CD66a (biliary glycoprotein), carcinoembryonic antigen (CEA) group member 2 (CGM2), and nonspecific cross-reacting antigen (NCA) are members of the human CEA gene subgroup. We investigated a series of 11 colorectal carcinomas by Northern blot and isotopic in situ hybridization (ISH), demonstrating underexpression of CD66a and CGM2 in the majority of the carcinomas as compared with the normal mucosa, whereas NCA was overexpressed. ISH for CD66a and CGM2 mRNA revealed that large areas of the carcinomas remained without or with only faint hybridization signals. However, in every carcinoma, at least some positive foci were observed, indicating remaining cell populations that actively transcribe CD66a and CGM2. In contrast, ISH for NCA displayed strong and extensive autoradiographic signals. By analysis of step sections, foci of CD66a and CGM2 expression were shown to co-localize. Furthermore, these foci contained relatively few nuclei immunohistochemically positive for the proliferation-associated nuclear antigen Ki-67. Our data indicate a dysregulation of the three genes possibly with a common transcriptional control for CD66a and CGM2 and a different control for NCA. The focal expression of CD66a and CGM2 could be interpreted as due to a focal, incomplete, and abortive differentiation or, alternatively, as a consequence of genetic heterogeneity with foci of slow-proliferating subclones.

Methods for detection of point mutations: performance and quality assessment. IFCC Scientific Division, Committee on Molecular Biology Techniques.

We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected, (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (f) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.

Expression of CD66a (human C-CAM) and other members of the carcinoembryonic antigen gene family of adhesion molecules in human colorectal adenomas.

Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis. Here we investigate the mRNAs of CD66a, CGM2, and NCA in 22 human colorectal adenomas and the respective normal mucosa specimens by Northern blots. The expression of both CD66a and CGM2 changed in a concomitant fashion. Using oligonucleotides specific for the N-terminal domains, two CD66a transcripts 3.9 and 1.5 kb in size were identified. These showed a greater than 50% down-regulation in 20 of 22 and 18 of 22 adenomas, respectively. Reduction of the CGM2 message was observed in 21 of 22 cases. Complete or near-complete losses of the CD66a 3.9-kb mRNA and the CGM2 message were found in 13 of 22 and 15 of 22 of the tumors, respectively. The medians of CD66a and CGM2 expressions were between 0.3 and 0.0, respectively. The tumor:normal ratio of NCA mRNA expression was increased up to 2.4-fold in 11 of 22 adenomas. Altogether, these results compare well to the changes reported previously for colorectal carcinomas. The high frequency and early appearance of dysregulation of members of the carcinoembryonic antigen family during colorectal tumorigenesis suggests that these changes may be important for the development of the malignant phenotype.

Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR.

Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkins lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene.

CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.

CD66a, also called biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and of the immunoglobulin superfamily. CD66a is the human homologue of Cell-CAM, a well-defined cell adhesion molecule of the rat. In the present study a monoclonal antibody specific for CD66a was used to locate CD66a in human tissues. CD66a is expressed in epithelia, in certain endothelia, and in cells of the myeloid lineage. Hepatocytes were stained along the bile canaliculi. A characteristic apical membranous staining was observed in enterocytes, superficial absorptive cells of the colon, in the epithelia of esophageal and Brunner's glands, bile ducts and gallbladder, pancreatic ducts, proximal tubules of the kidney, prostate, endometrium, and mammary ducts. Selective staining of endothelia was present in glomeruli and vasa recta of the kidney, small placental vessels, adrenal sinusoids, endometrium, the prostate. Among the cells of the myeloid lineage, granulocytes and myelocytes were positive. The expression of CD66a by human cells and tissues is well comparable with the expression reported for Cell-CAM, the rat counterpart of CD66a. The wide tissue distribution of CD66a indicates that CD66a is a prominent human adhesion molecule.

Tumour diagnosis by PCR-based detection of tumour cells.

Tumour cells shed from solid primary tumours can be detected by the polymerase chain reaction (PCR) based on the selective amplification of mutated tumour genes or of genes expressed in a tissue specific manner. When tumour specific alterations are amplified, few tumour cells can be detected in excess of normal cells derived from the same tissue. Thus, malignant cells can be detected specifically in pancreatic juice, stool, urine, and sputum. Here we describe the adaptation of the mutant enriched PCR in conjunction with the introduction of artificial primer mediated restriction sites to the selective amplification of mutant K-ras genes in stool samples from patients with colorectal carcinomas. In reconstitution experiments, down to 10 colorectal carcinoma cells could be detected in 100 mg of stool. For the diagnosis of micrometastatic disease, a sensitive and specific technique was established based on the reverse transcription of mRNA specific for the carcinoembryonic antigen followed by the amplification of the cDNA (RT-PCR). Attempts to establish a specific RT-PCR for cytokeratin-18 failed because of the existence of at least one processed pseudogene.

CGM2, a member of the carcinoembryonic antigen gene family is down-regulated in colorectal carcinomas.

We have determined the precise chromosomal location, the exon structure, and the expression pattern of CGM2, a member of the carcinoembryonic antigen (CEA) gene family. CGM2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT/PCR) from the colon adenocarcinoma cell line, LS174T. A defective exon is missing from this cDNA clone, leading to a novel domain organization for the human CEA family with two immunoglobulin-like domains. The derived C-terminal domain predicts that the CGM2 protein is membrane-bound through a glycosyl phosphatidylinositol anchor. RT/PCR analyses identified CGM2 transcripts in mucinous ovarian and colonic adenocarcinomas as well as in adjacent colonic tissue, but not in other tumors including leukocytes from six chronic myeloid leukemia patients. Thus, unlike several other family members, CGM2 is not expressed in granulocytes but reveals a more CEA-like expression pattern. Northern blot analyses identified a 2.5-kilobase CGM2 mRNA that is strongly down-regulated in colonic adenocarcinomas compared with adjacent colonic mucosa, suggesting a possible tumor suppressor function. In addition, a 3.2-kilobase transcript was observed in a number of colon tumors that is not detectable in normal colonic tissue. This mRNA species could represent a tumor-specific CGM2 splice variant.