HIF-1alpha determines the metastatic potential of gastric cancer cells.

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1alpha has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1alpha for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1alpha for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1alpha showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1alpha-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1alpha-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1alpha for migration, invasion and adherence argues for a pivotal role of HIF-1alpha in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1alpha-inhibiting substances in clinical treatment studies of advanced gastric cancer.

Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression.

Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R(0)-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor.

Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial.

PURPOSE: Metastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer. METHODS: Patients with histologically confirmed liver metastases from colorectal cancer were randomised to the vaccination or control group. After complete resection of liver metastases, patients randomised to the vaccination group received six doses of Newcastle disease virus (NDV) infected autologous tumour cell vaccine (ATV-NDV). The primary end-point was overall survival, secondary end-points were disease-free survival and metastases-free survival. RESULTS: Fifty-one patients were enrolled in the study with 50 patients available for analysis. The follow-up period was 116.1 +/- 23.8 month in the vaccination arm and 112.4 +/- 18.5 month in the control group. In the total patient group, no differences in the primary and secondary end-points were detected. Most interestingly, subgroup analysis revealed a significant advantage for vaccinated colon cancer patients with respect to overall survival [hazard ratio: 3.3; 95%, confidence interval (CI): 1.0-10.4; P = 0.042] and metastases-free survival (hazard ratio: 2.7; 95%, CI: 1.0-7.4; P = 0.047) in the intention-to-treat analysis. CONCLUSION: Active specific immunotherapy in unselected colorectal cancer patients was not effective for prevention of recurrent metastatic disease. However, in colon cancer patients, ASI with ATV-NDV appears to be beneficial prolonging overall and metastases-free survival.

Presence of mature DC-Lamp+ dendritic cells in sentinel and non-sentinel lymph nodes of breast cancer patients.

AIM: Our study examined differences in the presence of mature, DC-Lamp+ DC in the SLN and non-SLN according to the extent of metastatic involvement. PATIENTS AND METHODS: Paraffin blocks of the SLN and non-SLN from patients with primary breast cancer who had undergone SLN biopsy and axillary dissection were separated into three groups: (Group A) no tumor cell involvement in the SLN and non-SLN; (Group B) isolated tumor cells or micrometastases in the SLN, and tumor cell-free non-SLN; and (Group C) macrometastases in the SLN. One section of all the SLN and non-SLN was examined with immunohistochemistry using an anti-DC-Lamp-antibody. The densest area occupied by the DC-Lamp+ cells on each slide was quantified and recorded by an electronic imaging system. In this regard, the SLN and non-SLN were compared within the patients of each group using the Wilcoxon signed rank-test (p<0.05). RESULTS: One hundred and fourteen SLN and 1258 non-SLN from 79 patients were examined. A significantly larger area was occupied by the DC-Lamp(+) cells in the SLN compared to the non-SLN in Groups A (p=0.024) and B (p=0.009), whereas no significant difference was found within Group C (p=0.107). CONCLUSIONS: This study suggests that the DC-dependent immune response is altered during the process of metastasis formation and is primarily activated before and during formation of micrometastasis.

Markers of tumour angiogenesis and tumour cells in bone marrow in gastric cancer patients.

AIMS: Vascular endothelial growth factors VEGF-A, VEGF-C and VEGF-D are considered to be potentially angiogenetic and lymphangiogenetic. "Minimal residual disease" is responsible for cancer progression and recurrence. In this study, we investigated the relation between expressions of VEGF-A, VEGF-C and VEGF-D in gastric cancer tissue and the presence of tumour cells in bone marrow. METHODS: A total of 50 resected primary gastric adenocarcinomas, 44 non-cancerous gastric mucosa and 36 lymph node metastases were analyzed by immunohistochemistry for VEGF-A, VEGF-C and VEGF-D. The specimens used were drawn from a previous study cohort, where the presence of ITC in bone marrow was confirmed with immunohistochemical assay with cytokeratin (CK)-18. RESULTS: The levels of expression of VEGF-A, VEGF-C and VEGF-D were highest in tumour (p < 0.001), and the level in lymph node metastases was significantly higher (p < 0.01) than in mucosa. The expression of VEGF-A was correlated significantly with venous tumour invasion (p < 0.05) and the presence of tumour cells in bone marrow (p < 0.05). Tumours expressing high levels of VEGF-D showed significantly advanced stages of tumour infiltration (p < 0.05) and lymph node metastasis (p < 0.01). CONCLUSIONS: VEGF-A is a significant marker for the presence of tumour cells in the bone marrow of gastric cancer patients. Our results confirm VEGF-D as a predictor for the lymphatic spread of tumour cells. Therefore, the route of metastatic spread of gastric cancer could be determined, at least in part, by the profile of VEGF family members expressed in the primary tumour of gastric cancer patients.

The location of small tumor deposits in the SLN predicts Non-SLN macrometastases in breast cancer patients.

AIMS: The extent to which the location of micrometastases (MIC) or isolated tumor cells (ITC) in sentinel lymph nodes (SLNs) is correlated with the risk of downstream metastases is still unknown. This study examined this issue and compared the impact of MIC/ITC location with other established risk factors. METHODS: Paraffin slides of SLNs with MIC/ITC-involvement obtained from 68 breast cancer patients were evaluated for MIC/ITC location, lesion size, and various SLN morphologic features. These parameters, together with demographic data and primary tumor characteristics, were analyzed using univariate and multivariate analysis to determine their association with the presence of downstream macrometastases in Non-SLN. RESULTS: Eighteen of 68 patients with MIC (n=37) or ITC (n=31) had Non-SLN metastases. After multivariate analysis, the location of MIC/ITC in the SLN (parenchyma vs. sinus/vessel) had the strongest association with the presence of Non-SLN macrometastases (p<0.0001), followed by the pT-category (p=0.008). Sixteen of 18 patients with parenchymal involvement but only 2 of 31 without parenchymal involvement had Non-SLN macrometastases. The metric size of the primary tumor and the estrogen receptor status were significantly associated only on univariate analysis (p=0.041, 0.034), whereas the correlation to the size classification for tumor cell deposits (MIC vs. ITC) was not significant (p=0.077). CONCLUSIONS: The results indicate that lesion location is an important predictor of Non-SLN-macrometastases. This finding may simplify the decision for axillary treatment in patients with small tumor deposits in the SLN.

[Minimal residual tumor in gastrointestinal carcinoma. Relevance to prognosis and oncologic surgical consequences]. / Minimalresiduale Tumorerkrankung bei gastrointestinalen Karzinomen. Prognoserelevanz und onkologisch-chirurgische Konsequenzen.

Isolated tumor cells as a consequence of minimal residual disease are often not detectable by routine diagnostic procedures. However, before or after surgery, isolated tumor cells in lymph nodes, the peritoneal cavity, blood, or bone marrow can frequently be identified by immunohistochemical or molecular methods. Failure to reveal the presence of such cells results in under-staging of tumor patients and may constitute the source of unexpected tumor recurrence after radical surgery. These facts emphasize the importance of isolated tumor cells at least as a surrogate marker. The frequency of appearance of isolated tumor cells in different organ systems also depends on the type of primary tumor. Developments in modern detection methods have led to increasing sensitivity but at the expense of specificity. Isolated tumor cells demonstrate remarkable heterogeneity with respect to proliferative potential and tumorigenicity. This characteristic is also reflected by a striking variability in the expression of various genes conditioning the aforementioned biological behavior. Unfortunately there is also remarkable heterogeneity in methods used for sampling and processing patient material as well as for the enrichment and detection of isolated tumor cells. Despite the ongoing controversies concerning detection methods and biological significance of isolated tumor cells, several clinical trials providing data supporting the prognostic relevance of minimal residual disease should also be considered for gastrointestinal carcinoma. In future this finding should be integrated in the planning of trials in surgical oncology, and "minimal residual disease" should receive stronger attention as a stratification criterion in such clinical studies.

Glycosyltransferase expression in human colonic tissue examined by oligonucleotide arrays.

For an understanding of tumor-related alterations of the complex carbohydrate pattern of carcinomas, it is indispensable to monitor the expression profile of the various glycosyltransferases. The objective of this contribution was to perform an evaluation of the usefulness and the limits of the microarray approach for the identification of enzymes responsible for carbohydrate synthesis with differential expression in carcinomas. Expression profiles of colonic carcinomas were studied by oligonucleotide arrays using a novel strategy: colonic tissue of healthy individuals was compared with early staged colonic carcinomas; 'pure' cell populations were obtained by laser microdissection; RNA samples for hybridization with the oligonucleotide arrays were prepared by in vitro transcription without additional amplification. Expression of 39 glycosyltransferases and of 10 sulfotransferases in colonic tissues was analyzed by Affymetrix GeneChip technology. GeneChip analysis proved the high expression level of ST6Gal-I, beta4Gal-TI, II and III, GalNacT-1, FT-III and showed that ST3Gal-IV was the most abundantly expressed enzyme in healthy tissue. The strong overexpression of FT-VI in healthy tissue has not been described so far, as well as the upregulation of FT-VIII and downregulation of GnT-I in carcinoma tissue. Quantitative RT-PCR confirmed that FT-VI expression was significantly enhanced in healthy tissue. On the other hand, GeneChip analysis failed to detect any expression of GnT-III and GnT-V as well as of ST3Gal-I and ST3Gal-II, although these sequences could be amplified from the samples used for microarray analysis. According to our restricted analysis of only those 39 glycosyltransferases present on the GeneChip U95A, alterations of sialyltransferases ST6Gal-I, ST3Gal-IV, of fucosyltransferases FT-VI, FT-III, and probably FT-VIII, of GalNacT-I, and of beta4GalT-II seem to be of relevance for the aberrant biosynthesis of membrane-bound carbohydrates during colonic carcinogenesis and metastasis.

A rapid bioinformatic method identifies novel genes with direct clinical relevance to colon cancer.

Identifying genes whose differential expression affect the survival of patients after primary tumor surgery is a major aim of clinical cancer research. To address this issue we combined rapid bioinformatic search algorithms with quantitative RT-PCR in a panel of clearly defined cases of colorectal carcinomas with detailed patient histories. Search algorithms were written that identified Expressed Sequence Tags (ESTs) from the Unigene EST collection of putative open reading frames (ORFs). Expression ratios of healthy to cancerous tissue of each Unigene ORF were calculated. The first 35 candidates arising from bioinformatic searches were examined for mRNA expression in a panel of 20 well documented cases of colon cancer. Four of these 35 genes showed significant correlations with histopathological parameters. Therefore, their expression was further analysed by quantitative RT-PCR in a larger patient cohort. Kaplan-Meier/log rank statistical tests of up to 49 patients in three of the four genes demonstrated significant association of gene expression with poor survival. All four genes demonstrated a strong association with metastatic tumor progression. Expression of the genes was localized to epithelial cells by in-situ hybridization.

Overexpression of sialyltransferase CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-Sialyltransferase is related to poor patient survival in human colorectal carcinomas.

Thomsen-Friedenreich (TF)-related blood group antigens, such as TF, Tn, and their sialylated variants, belong to a family of tumor-associated carbohydrates. The aim of the present study was to examine tumor-associated alterations of glycosyltransferases involved in the biosynthesis of the TF glycotope in colorectal carcinomas. To this end, glycosyltransferase expression was examined in 40 cases of colorectal carcinoma specimens classified according to the WHO/Union International Contre Cancer guidelines and in "normal" mucosa of the same patients. Occurrence of TF glycotope was examined by immunohistochemistry with the monoclonal antibody A78-G/A7. Expression of sialyltransferases CMP-sialic acid:Galbeta1,3GalNAc-R alpha3-sialyltransferase I and II (ST3Gal-I and ST3Gal-II) and CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-sialyltransferase (ST6GalNAc-II) and of core 2 beta1,6-N-acetylglucosaminyltransferase was determined by reverse transcription-PCR in the same cryostat sections used for immunohistochemistry. Additionally, alpha2,3-sialyltransferase enzyme activity was studied in each of these tissues. The TF glycotope was detected in 7% of the normal mucosa, but in 57% of the carcinoma samples. Expression of alpha2,3-sialyltransferases ST3Gal-I, ST3Gal-II, and enzyme activity of alpha2,3-sialyltransferase was significantly increased (P < 0.001) in carcinoma specimens compared with normal mucosa. ST3Gal-I mRNA expression was significantly increased (P = 0.05) in cases showing invasion of lymph vessels. Expression of ST6GalNAc-II was significantly increased (P = 0.04) in cases with metastases to lymph nodes along the vascular trunk. Moreover, ST6GalNAc-II expression provides an prognostic factor for patient survival (log rank, P = 0.02). In an attempt to study the functional relevance of the glycosyltransferases for TF biosynthesis, SW480 colorectal cells were transfected with each of the enzymes, and cell surface expression of the TF glycotope was examined by flow cytometry. The presence of TF was not altered by transfection of the cells with either sialyltransferase ST3Gal-I or ST3Gal-II. However, successful transfection with core 2 beta1,6-N-acetylglucosaminyltransferase led to reduced expression of TF. In contrast, increased cell surface expression of TF was found after ST6GalNAc-II transfection. Thus, expression of TF on the cell surface of SW480 colorectal carcinoma cells depends on the ratio of core 2 beta1,6-N-acetylglucosaminyltransferase and ST6GalNAc-II. Earlier immunohistological studies demonstrated that TF is a prognostic factor for patient survival. Our results suggest that sialyltransferase ST6GalNAc-II is of crucial relevance for the prognostic significance of TF.

Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area.

The mediation of cAMP effects by specific pools of protein kinase A (PKA) targeted to distinct subcellular domains raises the question of how inactivation of the cAMP signal is achieved locally and whether similar targeting of phosphodiesterases (PDEs) to sites of cAMP/PKA action could be observed. Here, we demonstrate that Sertoli cells of the testis contain an insoluble PDE4D3 isoform, which is shown by immunofluorescence to target to centrosomes. Staining of PDE4D and PKA shows co-localization of PDE4D with PKA-RIIalpha and RIIbeta in the centrosomal region. Co-precipitation of RII subunits and PDE4D3 from cytoskeletal extracts indicates a physical association of the two proteins. Distribution of PDE4D overlaps with that of the centrosomal PKA-anchoring protein, AKAP450, and AKAP450, PDE4D3, and PKA-RIIalpha co-immunoprecipitate. Finally, both PDE4D3 and PKA co-precipitate with a soluble fragment of AKAP450 encompassing amino acids 1710 to 2872 when co-expressed in 293T cells. Thus, a centrosomal complex that includes PDE4D and PKA constitutes a novel signaling unit that may provide accurate spatio-temporal modulation of cAMP signals.

Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases.

BACKGROUND/AIMS: Biosynthesis of carbohydrate structures is tissue specific and developmentally regulated by glycosyltransferases such as fucosyltransferases, sialyltransferases, and N-acetylgluco- saminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumour subpopulations with different adhesion properties. Therefore alterations in glycosyltransferase mRNA expression in colorectal carcinomas were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Colorectal carcinoma specimens were classified and characterised according to the WHO/UICC system. Expression of fucosyltransferases FT-I, FT-III, FT-IV, FT-V, FT-VI, and FT-VII, sialyltransferases ST3Gal-I, ST3Gal-III, ST3Gal-IV, and ST6Gal-I, beta1,4-galacto- syltransferase, and beta1,6-Nacetylgluco- saminyltransferase V (GNT-V) was screened simultaneously in extracts of 22 homogenised tumour specimens by RT-PCR and compared with corresponding mucosa from each patient. Also 12 adenomas and 17 liver metastases of colorectal carcinomas were examined. RESULTS: GNT-V expression was enhanced in colorectal adenomas (p = 0.039), carcinomas (p<0.001), and liver metastases of colorectal carcinomas (p<0.001). Also, expression of fucosyltransferase FT-IV was increased in colorectal adenomas (p = 0.039) and carcinomas (p<0. 001). In addition, fucosyltransferase FT-I (p<0.001) and sialyltransferases ST6Gal-I (p = 0.004) and ST3Gal-III (p = 0.001) showed increased expression in carcinoma specimens. On the other hand, fucosyltransferase FT-III was less abundantly expressed in carcinomas exhibiting distant metastases (p = 0.046) and in highly invasive tumours (p = 0.041). CONCLUSIONS: Glycosyltransferase mRNA expression is significantly altered in colorectal adenomas and carcinomas isolated from surgical specimens. RT-PCR determination of specific glycosyltransferases may be helpful for earlier detection of carcinomas and for tumour prognosis.

Altered mRNA expression of glycosyltransferases in human gastric carcinomas.

Biosynthesis of carbohydrate structures is tissue-specific and developmentally regulated by glycosyltransferases like fucosyl-, sialyl- and N-acetylglucosaminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties. The aim of this contribution was to directly compare mRNA expression of several glycosyltransferases in surgical specimens of gastric carcinomas. Carcinoma specimens were classified and characterized according to the WHO/UICC system. In each case, the expression of 12 glycosyltransferase enzymes was studied simultaneously by RT-PCR. For semi-quantitative analysis, amplification of the sample sequence was compared with that of beta-actin, co-amplified within the same tube. Expression of N-acetylglucosaminyltransferase V in gastric carcinomas was significantly enhanced compared to normal tissue. Also, expression of sialyltransferase ST3Gal-IV and fucosyltransferase FT-IV was significantly enhanced in carcinoma tissue. No significant differences in glycosyltransferase expression were found in samples positive for Helicobacter pylori or between the different gastric regions. Thus, carcinogenesis is characterized by specific alterations in mRNA expression of several glycosyltransferases. Future studies will show whether RT-PCR detection of the expression of these enzymes could be helpful for prognostic purposes.

Preparation and characterization of differently aggregated colorectal carcinoma cell subpopulations from surgical specimens.

The loss of intercellular adhesion within the primary tumor is one of the key events leading to metastasis. Although a number of adhesion molecules involved in intercellular adhesion have been described in experimental systems, the clinical relevance of many of these molecules still has to be determined. We tried to assess the contribution of membrane-bound carbohydrates and of E-Cadherin, CEA, and Sia-LeA for intercellular adhesion of cells isolated from colorectal carcinoma tissue directly obtained from the surgeon. A subpopulation of nonaggregating cells was prepared by means of slowly passing of freshly isolated cells through a series of sieves with decreasing mesh widths. Nonaggregating cells differed mainly in two aspects from aggregated cells: (i) determination of lectin binding and of specific sialytransferase activities revealed enhanced alpha2,6-sialylation of nonaggregating cells, and (ii) staining with specific antibodies documented a loss of E-Cadherin reactivity of such cells. An enhanced activity of beta-galactoside alpha2,6-sialytransferase (ST6Gal 1) was found in metastasizing colorectal carcinomas; however, its biological function has to be shown. Our results suggest that ST6Gal 1 is responsible for reduced homotypic aggregation of colorectal carcinoma cells and may thus facilitate the release of single cells from the primary tumor.

[Significance of alpha 2,6-specific sialyltransferase ST6N and alpha 2,3-specific sialyltransferase ST3N in stomach carcinoma]. / Bedeutung der alpha 2,6-spezifischen Sialyltransferase ST6N und der alpha 2,3-spezifischen Sialytransferase ST3N beim Magenkarzinom.

The objective of this prospective study was to investigate the role of sialytransferase activities in patients with gastric cancer. In patients with gastric cancer we observed a significant correlation between sialyltransferase ST6N levels and survival after a median follow up of one year. High ST6N levels in the tumor and the surrounding normal mucosa were associated with poor prognosis. The results of this pilot study encourage further evaluation of sialyltransferase in patients with gastric cancer.

Inhibition of Gal beta1, 4GlcNAc alpha2,6 sialyltransferase expression by antisense-oligodeoxynucleotides.

Treatment of human colorectal carcinoma cells HT29 with specific antisense oligodeoxynucleotides led to a decreased Gal beta1,4GlcNAc alpha2,6 sialyltransferase activity on the level of protein expression as well as on the mRNA level. Antisense treatment did not effect cell viability or cell growth. Oligodeoxynucleotides which were complementary to the region upstream of the initiation codon were particularly effective in inhibition of enzyme expression. No such inhibition was found by treatment of cells with oligodeoxynucleotides complementary to the region downstream of the initiation codon or by treatment of cells with scrambled controls or sense oligodeoxynucleotides.

Colon carcinoma glycoproteins carrying alpha 2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-Tn antigen.

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of alpha 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the alpha 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for alpha 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.

Affinity purification of monospecific antibodies from polyclonal sera as a means for the identification of differentially expressed genes and proteins.

The identification and characterization of genes and proteins that are either cell-type specific or exhibit an otherwise spatially or temporally restricted expression pattern is a crucial step toward understanding the complex interactions between the different cell types in an organism. We demonstrate here that monospecific polyclonal antibodies purified from rabbit antisera by a simple Western blotting and reelution technique can be successfully used for the detection of such proteins, as well as the corresponding genes by screening cDNA expression libraries. This strategy is discussed as a supplement to other approaches, such as the generation of tissue-specific monoclonal antibodies and the "subtractive hybridization technique."

Different sialyltransferase activities in human colorectal carcinoma cells from surgical specimens detected by specific glycoprotein and glycolipid acceptors.

The amount and type of sialylation of tumor cell membranes depends on the activity of a number of different sialyltransferase enzymes. For the detection of specific activities in human colorectal carcinoma tissue several glycoprotein and glycolipid acceptors were used: desialylated fetuin, alpha 1-acid glycoprotein, beta 2-glycoprotein I, ovine submaxillaris mucin, and the gangliosides GM1, GM2, GM3 and GD1a. Because of their possible relevance for metastasis, precursors of Le(a) and Le(x) antigens, too, were employed, namely neoglycolipids produced by coupling LcOse4 or NeoLcOse4 oligosaccharides to L-alpha-phosphatidyl-ethanol-amine-dipalmitoyl. Our data indicate that human colorectal tumor tissue contains two highly active sialyltransferase enzymes, which are only weakly expressed in normal mucosa. These are a N-glycan-specific alpha 2,6-sialyltransferase, which was significantly increased in metastasizing tumors, and a Gal beta 1,3Gal-NAc-specific sialyltransferase, which was increased in tumors of early stages. A shift to enhanced alpha 2,6-sialylation of membrane glycoproteins during carcinogenesis was demonstrated by lectin ELISA analysis of magneto-bead separated tumor cells. Quantitative determination of specific sialyltransferase activities may be a sensitive tool for detection and monitoring of colon carcinoma.

Enhanced activity of CMP-neuAc:Gal beta 1-4GlcNAc:alpha 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients.

The activity of sialyltransferases with different linkage specificities, of a Gal beta 1-4GlcNAc:alpha 2,6-sialyltransferase and a Gal beta 1-4GlcNAc:alpha 2,3-sialyltransferase, was studied in human colorectal tumor tissue from surgical specimens, normal mucosa, liver and liver metastases, and serum of patients suffering from colorectal carcinomas. While alpha 2,3-specific activity was equally high in tumor and mucosa samples, the activity of the alpha 2,6-specific enzyme was increased in tumor tissue and particularly in metastasizing tumors. Also, compared to healthy individuals, serum of patients suffering from metastasizing tumors contained a significantly higher activity of the alpha 2,6-specific enzyme. These results demonstrate that specific sialyltransferase isoforms are expressed in metastasizing tumors and that determination of such isoforms may be a new means for tumor detection and monitoring.