Detection of tumor cells in the portal and peripheral blood of patients with colorectal carcinoma using competitive reverse transcriptase-polymerase chain reaction
Abstract:BACKGROUND
In spite of many reports, it remains unclear whether the presence of tumor cells in circulating blood flow predicts a poor prognosis.
METHODS
Competitive seminested reverse transcriptase‐polymerase chain reaction (RT‐PCR), a technique for the quantitative detection of tumor cells, was applied to detect the presence of tumor cells in portal and peripheral blood samples from 121 patients with colorectal carcinoma and to clarify their clinical significance. This technique can detect one carcinoembryoni… Show more
“…The 7700 Sequence Detector System (Applied Biosystems) was used for Real-time quantitative RT-PCR (32,33). Briefly, each RT-PCR reaction mixture (50 μl) included 250 ng of total RNA, 25 μl of Taqman Universal PCR Master Mix (Applied Biosystems), 0.5 μl of forward and reverse primer (10 μM), and 1.0 μl of corresponding Taqman probe (5000 nM).…”
Abstract. System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na + -independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 -1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14 C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.
“…The 7700 Sequence Detector System (Applied Biosystems) was used for Real-time quantitative RT-PCR (32,33). Briefly, each RT-PCR reaction mixture (50 μl) included 250 ng of total RNA, 25 μl of Taqman Universal PCR Master Mix (Applied Biosystems), 0.5 μl of forward and reverse primer (10 μM), and 1.0 μl of corresponding Taqman probe (5000 nM).…”
Abstract. System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na + -independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 -1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14 C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.
“…This observation may partially explain the negative conclusions reached by other authors, who suggested that the presence of circulating tumor cells might be of little value as a prognostic factor, because they appeared not to be associated with conventional prognostic factors, namely the presence of metastatic disease. 20 The findings here of a significant association between increased sE-selectin and positive CEA mRNA-expressing cells in the peripheral blood of patients with low-grade inflammatory status (as evidenced by increased levels of TNF-a) fuel the hypothesis that circulating tumor cells may, indeed, represent a prognostic factor, but only in those patients in whom they are capable of triggering the molecular changes demonstrated in the studies cited above, 6,7 and suggest that circulating tumor cells might be responsible for macrophage activation in vivo, release of proinflammatory cytokines, and up-regulation of vascular E-selectin, which can be detected in its soluble form on shedding from the endothelial cell surface. This hypothesis is also consistent with the finding that the highest sEselectin levels were detected in patients highly expressing CEA mRNA and with positive TNF-a levels compared with patients with negative levels of both variables.…”
BACKGROUND: This study analyzed the possible prognostic value of presurgical serum soluble (s)E-selectin levels and/or carcinoembryonic antigen (CEA) mRNA positivity in predicting the disease-free survival of colorectal cancer (CRC) patients. METHODS: CEA mRNA (obtained from blood-borne cells by reverse transcriptase-polymerase chain reaction [RT-PCR]), tumor necrosis factor-a (TNF-a), and sE-selectin levels were analyzed in blood samples obtained from 78 patients with primary (n ¼ 62) or recurrent (n ¼ 16) CRC, 40 patients with benign colorectal (CR) diseases, and 78 controls. RESULTS: CEA mRNA positivity by RT-PCR was significantly associated with advanced stage (P < .05). Median baseline sE-selectin levels were higher in patients with CRC (43 ng/mL) compared with controls (36 ng/mL) or patients with benign CR diseases (31 ng/mL, P < .001). These were significantly associated with CEA mRNA positivity by RT-PCR (P < .05). Multivariate analysis by forward stepping showed that elevated TNF-a (P ¼ .001) and CEA mRNA positivity by RT-PCR (P ¼ .0001) were independent predictors of elevated baseline sE-selectin levels. Positive presurgical sE-selectin levels were associated with an increased recurrence rate compared with patients with low levels of this molecule (P < .001). Positivity for both CEA mRNA and sE-selectin had a negative prognostic impact, with a 5-year recurrence-free survival rate of 51% compared with 95% of patients with negative parameters (P < .05). CONCLUSIONS: Detection of presurgical serum sE-selectin levels and CEA mRNA-positive blood-borne cells in CRC patients might provide useful prognostic information in terms of recurrence-free survival, either alone or in combination, and may help in the choice of more aggressive treatment and/or more strict follow-up procedures in high-risk patients.
“…The association of tumorspecific mRNA detection in the peripheral blood with the various clinicopathological parameters has been a matter of significant debate. There is reported evidence to a significant association of disseminated cancer cell detection to advance stage and lymph node metastasis [21,45]. On the other hand, there are studies that no correlation can be detected between the CEA mRNA detection and the clinicopathological characteristics of the disease [7].…”
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