A novel role for DYX1C1, a chaperone protein for both Hsp70 and Hsp90, in breast cancer

Aims  With three consecutive tetratricopeptide repeat (TPR) motifs at its C-terminus essential for neuronal migration, and a p23 domain at its N-terminus, DYX1C1 was the first gene proposed to have a role in developmental dyslexia. In this study, we attempted to identify the potential interaction of DYX1C1 and heat shock protein, and the role of DYX1C1 in breast cancer. Main methods  GST pull-down, a yeast two-hybrid system, RT-PCR, site-directed mutagenesis approach. Key findings  Our study initially confirmed DYX1C1, a dyslexia related protein, could interact with Hsp70 and Hsp90 via GST pull-down and a yeast two-hybrid system. And we verified that EEVD, the C-terminal residues of DYX1C1, is responsible for the identified association. Further, DYX1C1 mRNA was significantly overexpressed in malignant breast tumor, linking with the up-regulated expression of Hsp70 and Hsp90. Significance  These results suggest that DYX1C1 is a novel Hsp70 and Hsp90-interacting co-chaperone protein and its expression is associated with malignancy. Yuxin Chen, Muzi Zhao, Saiqun Wang contributed equally to this work.     

No association between <Emphasis Type="Italic">MDR1</Emphasis> (<Emphasis Type="Italic">ABCB1</Emphasis>) 2677G>T and 3435C>T polymorphism and sporadic colorectal cancer among Bulgarian patients

Purpose Variation in genetic factors together with xenobiotic exposure may result in increased risk of colorectal cancer. The P-glycoprotein (P-gp) is highly expressed in the apical membrane of enterocytes, where it pumps xenobiotics from the enterocytes back into the intestinal lumen. Thus, polymorphisms that reduce the activity of the MDR1 (ABCB1) efflux pump are potential risk factors for colorectal carcinogenesis. The aim of the present study is to genotype the MDR1 2677G>T (rs2032582) and 3435C>T (rs1045642) polymorphism in patients with colorectal cancer and controls and to identify a possible association between individual genetic variation and susceptibility to colorectal cancer. Methods In the present study, 146 Bulgarian patients with sporadic colorectal cancer and 160 healthy Bulgarian volunteers were evaluated for the two polymorphisms in MDR1. Polymorphisms were identified using rapid-cycle real-time amplification with allele-specific probes and subsequent melting curve analyses on a LightCycler™ (Roche Diagnostics, Mannheim, Germany). Results No differences were found between the frequencies of the two mutant alleles in the tumor tissue from the cases and lymphocytes from the controls [frequencies of 2677T: 43.5% in patients and 44.1% in controls; frequencies of 3435T: 48.3% in patients and 50.9% in controls (both P > 0.05)]. The MDR1 polymorphic sequence of the tumor tissue always matched that of normal intestinal tissue from the same patient. Consequently, genotyping of DNA from archived tumor tissues is a valid alternative to the use of leukocyte DNA. Conclusions The present study suggests that MDR1 2677G>T and 3435C>T polymorphism is not a risk factor for sporadic colon cancer among Bulgarians and that somatic mutation at these sites is not involved in the genesis of colon tumors. Further examination using larger number of samples must be necessary to reach to more reliable conclusions.     

Decreased Expression of Monocyte Chemoattractant Protein-1 Predicts Poor Prognosis Following Curative Resection of Colorectal Cancer

Purpose  The significance of monocyte chemoattractant protein-1 in colorectal cancer is not well understood. The aim of this study was to investigate the significance of monocyte chemoattractant protein-1 expression in colorectal cancer patients undergoing potentially curative surgery. Methods  We studied 101 colorectal cancer patients who underwent potentially curative surgery. The concentration of monocyte chemoattractant protein-1 in the tumor and normal mucosa were measured. The expression of monocyte chemoattractant protein-1 was also evaluated immunohistochemically. Results  The tissue concentration of monocyte chemoattractant protein-1 in the tumor was significantly higher than that in the normal mucosa. The decreased monocyte chemoattractant protein-1 cancer/normal ratio was associated with lymph node involvement and could predict poor prognosis. On univariate analysis, the decreased monocyte chemoattractant protein-1 ratio, carcinoembryonic antigen levels, and serosal invasion were the significant factors for poor prognosis. Multivariate analysis showed that monocyte chemoattractant protein-1 ratio was the only independent risk factor predictive of a poor prognosis. Immunohistochemically, monocyte chemoattractant protein-1 was expressed in the cytoplasm. Conclusion  The decreased monocyte chemoattractant protein-1 ratio was an independent factor predicting poor prognosis in patients undergoing potentially curative surgery. Monocyte chemoattractant protein-1 deficiency may present a new therapeutic approach for colorectal cancer.     

Cyclin-dependant kinase inhibitors CIP1 (p21) and KIP1 (p27) in ovarian cancer

Purpose: Deregulation of the cell cycle is one of the important prerequisites for cancer development. p21 and p27 are both universal inhibitors of cyclin-dependant kinases and can therefore influence cell cycle or tumor progression. The aim of this study was to determine the influence of p21 and p27 expression on survival and chemotherapy response. Methods: 165 patients with ovarian cancer have been examined for p21 and p27 expression by immunohistochemistry on formalin-fixed, paraffin-embedded tissue using the monoclonal primary antibody WAF1 (Oncogene Science) and KIP1 (Transduction Laboratories). Results: High p21 expression (>50%) correlates only with early tumor stage (P=0.04). There was no correlation found between p21 and p27 expression. Patients with high p27 expression (>25%) had a longer DFS (disease free survival) in both univariate and multivariate analysis (P=0.05 and P=0.043) than patients with low p27 expression. A longer overall survival (OS) could only be proven for the group of high p27 expression in univariate analysis (P=0.03). Conclusion: p27 is an independent prognostic factor for ovarian cancer for DFS though this was not true for OS.     

Membrane-type 1 matrix metalloproteinase mRNA expression in colorectal cancer

PURPOSE: Membrane-type matrix metalloproteinases are recently described proteolytic enzymes belonging to the matrix metalloproteinase family. Initial studies have indicated that membrane-type matrix metalloproteinases are involved in tumor invasion and metastasis. Membrane-type 1 matrix metalloproteinase is the first membrane-type matrix metalloproteinase to be described. The aim of this study was to investigate the expression of membrane-type 1 matrix metalloproteinase mRNA in colorectal cancer. METHODS: Samples were collected from surgical specimens of patients with colorectal adenocarcinoma and were immediately frozen in liquid nitrogen and stored at –80°C until processed. Both normal and cancer tissue was taken from each patient. TNM stage, tumor differentiation, mucin production, and vascular invasion were assessed. Northern blotting was used to quantify membrane-type 1 matrix metalloproteinase mRNA levels in the samples using a membrane-type 1 matrix metalloproteinase cDNA clone. X-ray film images were digitized and densitometry was used to quantify bands. All samples were normalized against 18S rRNA levels. Results are expressed as the ratio of cancer to normal tissue levels. Statistical analysis was performed using analysis of variance, withP<0.05 accepted as the level of significance. RESULTS: A total of 32 samples were prospectively analyzed. The correlation between TNM stage and increased expression of membrane-type 1 matrix metalloproteinase mRNA in cancer tissue over normal tissue is expressed in the mean ratio of cancer to normal tissue expression for Stages I through IV, respectively: 1.4 ± 0.2 (12 patients); 4.1 ± 2.6 (8 patients); 3.4 ± 3 (7 patients); and 4.5 ± 5 (5 patients). Stage I is significantly different from Stages II and IV (P<0.05). These preliminary results show an overall increasing trend in membrane-type 1 matrix metalloproteinase expression with increasing tumor stage. However, there was no correlation between membrane-type 1 matrix metalloproteinase expression and mucin production, degree of tumor differentiation, or vascular invasion. CONCLUSION: Preliminary results indicate that membrane-type 1 matrix metalloproteinase levels correlate with increasing tumor stage.Supported in part by a research grant from the David G. Jagelman memorial research fund.Read at the meeting of The American Society of Colon and Rectal Surgeons, San Antonio, Texas, May 2 to 7, 1998.     

ABCB1 gene polymorphisms and haplotype analysis in colorectal cancer

Purpose  The aim of this study was to determine the significance of three most common single-nucleotide polymorphisms (SNPs) of ABCB1 gene in the development of colorectal cancer and to estimate the influence of these SNPs to surviving patients' treatment combination adjuvant therapy 5-fluorouracil/leucovorin. Haplotype structure of ABCB1 was analysed, and degree of linkage disequilibrium (LD) between SNPs of ABCB1 was estimated. Materials and methods  Tumour specimens of 95 patients with colorectal cancer and blood samples of 95 healthy cases were studied. Genotyping of ABCB1 gene was performed by automated sequencing or polymerase chain reaction-restriction fragment length polymorphism method. Comparison of frequencies of alleles/genotypes/haplotypes between the studied group (colorectal cancer samples) and the control group (blood samples) were analysed. These results were correlated with the surviving patients after treatment of adjuvant chemotherapy. Results  Significant differences in ABCB1 _1236C>T (p = 0.00043) and ABCB1 _2677G>T/A (p = 0.04) genotype distribution and T₁₂₃₆ allele distribution (CT₁₂₃₆ or TT₁₂₃₆ vs CC₁₂₃₆; p = 0.0499, OR = 0.55, Fi–Yule coefficient = 0.14) were found. A strong LD between ABCB1 _1236C>T and ABCB1 _2677G>T/A SNPs (D′ = 0.621, r ² = 0.318) was detected. All SNPs were located in one haplotype block. There were significant differences in haplotype distributions between colorectal cancer patients and healthy population (p = 0.03). Significant differences in survival probability of colorectal cancer patients' treatment chemotherapy according to allele of ABCB1 _3435C>T was observed. Survival probability of patients with wild-type C₃₄₃₅ allele were higher than among patients without this allele (p = 0.04572). Conclusions  These results suggested that three studied SNPs of ABCB1 were located in one haplotype block. Differences in ABCB1 _1236C>T and ABCB1 _2677G>T/A genotypes and T₁₂₃₆ allele distribution between investigated populations indicate significant impact of these SNPs on risk of development of colorectal cancer. Polymorphism ABCB1 _3435C>T may be a prediction marker of cancer chemotherapy effectiveness. Differences in haplotype distributions between colorectal cancer patients and healthy population suggested that other potential SNPs, especially in regulatory region of ABCB1 gene, may influence P-glycoprotein expression and function.     

Tiam1、Rac1在评估大肠癌远隔脏器转移中的意义

目的:观察人大肠癌和癌旁正常大肠黏膜组织中T淋巴瘤侵袭转移诱导因子1(Tiam1)和RacGTP酶激活蛋白1(Rac1)的表达,并探讨其与大肠癌根治术后发生远隔脏器转移的相关性.方法:应用SP免疫组织化学法检测87例大肠癌及40例癌旁正常组织标本中Tiam1/Rac1的表达情况;应用竞争性RT-PCR检测上述标本中Tiam1 mRNA的表达情况;应用配体沉淀法检测上述标本中Rac1蛋白活性.结果:Tiam1/Rac1在大肠癌细胞胞质内呈阳性染色.Tiam1 mRNA在大肠癌中表达明显增强(0.6±0.02vs0.24±0.02,P<0.0005),并且在术后发生远隔转移患者中的表达要高于未发生远隔转移者(0.91±0.02vs0.52±0.02,P<0.0005).Rac1蛋白活性的表达情况与Tiam1一致,在大肠癌中表达高于正常黏膜(0.17±0.01vs0.07±0.05,P<0.0005),并且在有远隔转移的大肠癌组织中活性要高于无远隔转移者(0.25±0.02vs0.15±0.01,P<0.0005).对于不同分化程度的大肠癌组织,Tiam1/Racl的含量均没有统计学差异(0.63±0.04,0.60±0.04,0.57±0.04,P=0.613;0.18±0.06,0.17±0.05,0.15±0.05,P=0.558).结论:Tiam1/Racl是大肠癌根治术后患者发生远隔转移的正性因子,可以作为预测大肠癌根治术后患者发生远隔脏器转移的有效指标.     

葡萄糖醛酸转移酶1A基因的组织差异性表达及多态调节

目的从mRNA、蛋白质及细胞水平分析尿苷二磷酸葡萄糖醛酸转移酶（UGT）1A基因的组织差异性表达及多态凋节。方法采用逆转录聚合酶链反应分析结直肠癌组40例、正常人群肠道黏膜20例、正常肝组织10例的UGT 1A mRNA表达；免疫印迹检测各组UGT 1A蛋白表达；高效液相色谱法测定各组UGT 1A酶的催化活性。结果①结直肠癌组织中UGT 1A mRNA表达量明显低于其周围正常黏膜，而后者低于正常人群肠黏膜组织，正常人群结直肠黏膜中UGT 1A mRNA表达量总体低于肝组织，P〈0．01。结直肠癌组、正常人群结直肠黏膜组织呈现个体差异表达，而肝组织的表达较均质。②癌组织、癌周正常黏膜及正常人群肠道黏膜中UGT 1A各同工酶的表达例数均不相同。③UGT 1A蛋白表达在癌组织显著低于癌周正常组织，后者又低于正常人肠黏膜（P〈0．01），各组蛋白表达量亦各不相同。而肝组织呈均质表达。④癌组织的UGT 1A酶活性低于癌周正常黏膜．而后者的微粒体酶活性低于正常人群肠黏膜微粒体蛋白，P〈0．01。结论①UGT 1A基因位点呈组织差异性表达。②结直肠黏膜上皮UGT 1A基因位点在转录水平及功能水平均存在多态调节，不同个体对致癌物的易感性不同可能是这种差异表达的结果。     

Invasion and metastasis of liver cancer: expression of intercellular adhesion molecule 1

To study the relationship between intercellular adhesion molecule 1 (ICAM-1) and liver cancer metastasis and to find predicting factors that could indicate the growth and metastasis of liver cancer. Methods: ICAM-1 expression in fresh tissue of normal liver and hepatocellular cancer (HCC) was examined by immunoperoxidase staining. Serum soluble intercellular adhesion molecule 1 (sICAM-1) from patients with a benign HCC tumor, and the expression of ICAM-1 in the orthotopically transplanted LCI-D20 tumor of a nude mouse liver cancer metastasis model, and in human hepatoma, the tumor surrounding tissue and normal liver, was analyzed semiquantitatively by the immuno-dot blot method. Tissue ICAM-1 expression (mRNA level) was detected by Northern blotting. Results: ICAM-1 expression in LD1-20 D metastatic liver cancer had a positive correlation with tumor size and the time after implantation. It increased suddenly as metastasis occurred being 3.03 ± 0.51 before metastasis and 8.24 ± 0.95 after metastasis, P < 0.01, then remained high, appending on the number of sites involved (monosite metastasis 5.48 ± 0.49, multisite metastasis 10.05 ± 1.17, P < 0.05). All six cases of normal liver samples were negative in anti-ICAM-1 immunohistochemical staining, 80.0% (36/45) of the HCC showed some ICAM-1 expression. The rate of positive cells was a little higher in large tumors, tumors with an intact capsule and tumors with metastasis, but there was no significant difference. It was noticed that two cancer emboli also had high ICAM-1 expression. The ICAM-1 concentration in HCC (13.43 ± 0.09) was higher than that in tumor surrounding the liver (5.89 ± 0.17, P < 0.01) and that in normal liver (4.27 ± 0.21, P < 0.01). sICAM-1, like tissue ICAM-1, was higher in HCC patients than in patients (with benign liver tumor and normal controls. Both tissue ICAM-1 and sICAM-1 were higher in the metastasis group than in the group without metastasis (tissue ICAM-1 20.24 ± 0.30 vs 10.23 ± 0.12 P < 0.05; sICAM-1 12.18 ± 0.25 vs 9.77 ± 0.54 P < 0.05). Northern blot analysis revealed that ICAM-1 expression, as indicated by mRNA level, was also higher in HCC and in cancer emboli than in tumor surrounding liver and normal liver. Conclusions: Tissue ICAM-1 and serum sICAM-1 could indicate the stage of HCC, and the potential of hepatoma cells for invasion and metastasis. They may play an important role in the metastasis cascade. Received: 20 January 1998 / Accepted: 25 June 1998