| During recent years it has been realized that Tiam1-Rac1 pathway may take a part of catenating relay in the labyrinth-like network for intra-cell signaling transduction. Either tyrosine receptor kinase (Trk) family or the so called coreceptors, which deliver activating signals through non-receptor protein tyrosine kinases (PTK), triggers phosphorylation and activation of Tiam1. Besides these, affiliated receptors of Wnt proteins and the traditional extra-cellular matrix (ECM) -integrins perform their functions through Ras to activate Tiam1 and then Rac1 to regulate cell behaviors, whereas the reactive oxygen species(ROS) keeps Tiam1 active by the non-specific pattern of suppressing dephosphorylation process.As the professional guanine-exchange factor (GEF) of Rac1, Tiam1 is able to promote Rac1 activation both in vitro and in vivo by catalyzing GTP load on the substrate. The active Rac1 elicits a number of terminal kinases also by their phosphorylations on serine (S) and threonine(T) residues. Such kinases include p21AK for dynamic reorganization of actin cytoskeleton (DRAC), p38MAPK for transcription activation, and transcript factors NFκB, AP1 and STAT3 for direct transcription. The transcript factors translocate into caryon and initiate transcription as soon as the phosphorylation of themselves after binding to active Rac1.Tiam1-Rac1 signaling pathway has been reported to be involved in progress of cancer and be responsible for maligmant phynotypes of tumor cells such as invasion and metastasis. A lot of investingations have attributed these manifestations to the abnormal DRAC process in tumor cells, though it has been admitted that cancer belongs to such a kind of diseases that develop as the results of somatic (cell) mutations. In the dissertation, I describe aberrant activation of Tiam1 and Rac1 transcription in 66 cases suffering from gastric carcinomas, which is marked by obviously increased amount of the encoding sequences in the tumor tissues, and defined as super-overexpression for both the target genes. As what I have known based on searching in Pubmed, it may be the first sentence for linking tumor-related aberrant activation of transcription to pathogenesis of cancerous diseases.The SLN micrometastasis in gastric carcinoma analyzed by RTFQ-PCR for CK18 transcripts The majority of cancerous metastasis into lymphoid nodes could be found by routine examinations of pathological H-E staining, and the immunohistological techniques of avidin-biotin enzyme complex can even find out the micrometastasis composed of only a few carcinoma cells in a SLN. As the result, this kind of assays has been authorized in clinical application for diagnosis of carcinoma staging in some nations such as the United States. In spite of the much higher sensitivity, it might be probable to omit the real existence of micrometastasis in the SLNs analyzed, simply due to being not the sites cut where the micrometastatic tumor cells have harbored. On the basis of investigation for the correlation between the micrometastases of gastric carcinoma and the RT-PCR detections with cytokeratin 18 (CK18) as the hallmarker, we develop an objective and highly sensitive assay system of RTFQ-PCR to determine the copy numbers of CK18 transcripts in SLNs, whereby to identify micrometastases of epithelial carcinoma cells.We have constructed recombinant TA clones inserted with CK18 cDNA sequence, and used it as the standard to evaluate the target mRNA transcripts in SLNs of the patients suffering from gastric carcinomas. We assured that CK18 mRNAs as few as 10 transcripts were capable of being hitted by repeat assays in the RTFQ-PCR detection. The specificity for CK18 amplification was identified by the PCR product of expected sizes (-100bp) in agarose gel electrophoresis and of sequencing, and the profile of agarose gel electrophoresis was accounted for the quality control available in the RTFQ-PCR analysis.There were no positive detections of CK18 expression in the draining lymph nodes removed from 11 individuals with benign gastric illness and also, no negative for CK18 amplification in the pathologically diagnosed positive SLNs. Therefore, we are sure that the RTFQ-PCR examination be correct in terms of the results above. In our experiments, the recall rate for lymph node micrometastasis was significantly raised in gastric cacinoma by RTFQ-PCR. In the experiments including 66 cases of gastric carcinomas, we have found 15 patients with SLN micrometastasis in 24 individuals who had been diagnosed as negative according to LN pathology. The positive rate for micrometastasis reached 62.50% (15/24) in the cases enrolled in the study. It was indeed true no widely accepted common-norm for the relationship between quantifying the number of epithelial transcripts in LNs and tumor micrometastasis as yet, and an epidemiological survey was on the way, which was concerned with quantization of CK18 transcripts in LNs to recurrences and prognosis of gastric carcinoma, so that it was unable to reveal, practically, clinical significance of quantifying CK18 transcripts in LNs until now. On the other hand, I could at least define the measured CK18 transcripts beyond 10~4 in a LN as the occurrence of the viable metastatic cancer cells, abiding by recent achievements in micrometastasis as well as the definition for high-abundance of a given mRNA species in a single cell. In terms of the criterion, 13 cases in the 24 patients be considered as ones with active micrometastasis. We suppose that it may be meaningful for prolonging the tumor-free lifespan of patients, decreasing the probability for cancer recurrence and improving the comprehensive regimens to gastric carcinomas if the assay was clinnically used. We need a retrospective investigation later to point out the significance of the diagnosis.Gastric carcinoma-related aberrant activation for transcription of Tiam1 and Rac1 Tiam1 stands for T lymphoma invasion and metastasis inducing factor 1, and it has been believed to associate with in situ-invasion and metastasis of malignant cells since discovered. In order to exhibit the pathogenic significance of the signaling molecules in telephic neoplasts, we have for the first time analyzed the expressing levels of Tiam1 with an absolute quantification by the RTFQ-PCR analysis, which included a standard curve following statistic principles with recombinant Tiam1 TA clone as the standards, and with investigation for the sensitivity at 10 copies and the quality control parameter of the sole band of Tiam1 (and only 1 band for the quality control of the cDNA samples at 500 bp ofβ-actin to exclude possible contamination of genomic DNA in the samples). By using the detecting system and relative quantitation for Rac1 by RT-PCR, we analysed the clinico-pathological and possible pathogenic significance of the amounts of Tiam1 and Rac1. Gastric carcinomas were removed from the 66 patients, and both the cancerous tissue and the para-ventriculus tissue (3-5cm in the distance from carcinoma mass) were taken immediately after gastrectomy and stored in liquid nitrogen until arriving the lab and extracting the total RNA. 11 benign stomach tissues of ulcer were utilized as the negative control for the research. No sooner had the total RNA been extracted than the cDNA was prepared, and maintained at -76℃till used in PCR. In the study, we simultaneously measured Rac1 expressing by relative quantitation of the R values (by the formula R=ΔRac1/Δβ-actin).It has been found that the transcript amount of Tiam1 in the gastric cancer tissues varied among different origins and individuals. The expressing levels were related to invasive extent of the in situ carcinomas and (micro)metastasis in LNs, and consistent with the clinical TNM staging of the patients. Most importantly, the copy numbers of Tiam1 transcripts were correlated with degrees of cell differentiation of the carcinoma cells in tumor tissues significantly. Besides its relationships with the pathological and clinical manifestations, an obvious colinearity was found between Lg [Tiam1] and R values for Rac1. It was firstly proposed that the super-overexpression of both Tiam1 and Rac1 be considered as a definite sign that indicates the aberrant activation for the transcription in malignant tumors. It was shown in the study that (micro)metastasis of gastric carcinomas concerned super-overexpressions of Tiam1 and Rac1 closely. We postulated Tiam1 >10~4and Rac1 > 1.4 as their super-overexpressions and positive CK18 transcripts in LNs as micrometastasis(+) and found that (1) percentage for Tiam1 super-overexpression reached 90.48% (38/42) and 83.33% (35/42) for Rac1 in the patients with LN metastasis. The expressing levels of both were presented in a fashion of positive colinearity (r = 0.854 , P < 0.01). (2) 24 cases without pathological metastasis were found positive in micrometastasis by RTFQ-PCR and the recall factor was 62.50% (15/24) with LN positive ratio of 22.11%(44 positive in 199 LNs). A positive linear correlation was present for the expressing level of CK18 and either those of Tiam1 and Rac1. (3) only 9 of the 66 patients belonged to individuals without metastasis, for negative findings not only in pathology but also in RTFQ-PCR. There was no Rac1 expression in 7 of them, of whom 3 were no expression of Tiam1, 3 with Tiam1 less than 10 copies and another with Tiam1 copies at 1.05×10~4. The copy numbers of Tiam1 transcript were < 104 in the other 2 cases without metastasis but of rac1 expression positive. As the results, we concluded that an obvious concordance for their expressions existed among the 3 parameters (4) The 9 cases 77.78%(7/9) were all in clinical TNM I or II stages, indicating clinical significance for the quantitating examinations of Tiam1 and Rac1 expression. (5) it was exhibited that a closer concern was found between the expressing levels of Tiam1 and Rac1 by the statistically correlation of the sum of ranking analyses, and the changing trends of them were well accompanied with both the clinical and pathological parameters. Based on the observations and analyses above, I suppose that Tiam1-Racl signaling pathway play a role for promoting metastasis and progression of gastric carcinoma, it was possible that Tiam1, the specific GEF for Rac1, up-regulate the down-stream effectors through activating Rac1 to aggravate the carcinomas, and the super-overexpression of both Tiam1 and Rac1 may be the specific hallmarks for gastric carcinoma-associated abnormal activation of transcription.Rac1 variation in gastric carcinomas and the hypothesis for pathogenesis of the constitutively active Rac1: PCR-SSCP was conducted in order to find whether there were genetic variations in Rac1 sequence. We artificially selected 3 cDNA samples with the highest Rs in Rac1 RT-PCR and 11 ones with low level of Rac1 expression as the control in our studies. In the experiment, we identified the gene variation of Rac1 transcripts in the gastric carcinomas. Three cDNA samples with high Rs in RT-PCR showed the same position of band in the non-denatured PAGE uniformly, which differed from that of the control sample from 11 patients with the similar disease, but low levels of Rac1 expression, indicating the distinguishment of the cDNA from the cacinoma with high expression of Rac1, from those with low level of expressing. Based on these findings,we conclude that there is the variation of the enconding sequence for the gene in at least part of the patients with super-overexpression of Rac1. We assure that no such reports exist in research on gasric cacinoma according to searching in Pubmed, though the charactors of the variation remain undefined. Therefore, we suppose that the variation may be involved in somatic mutation of gasric cacinoma, and it may be of the pathogenic significance. It is concluded that the 3 cDNAs from the patients with gastric carcinomas exhibited the same characters of variation in their Rac1 gene. We suppose that variation in Rac1 encoding sequence occurs at least in a portion of patients with gastric carcinomas. As what I know, it is the first research that links variation in Rac1 mRNA to gastric carcinoma.In molecular genetics, a lot of work has indicated that the experimental mutations in Rac1 result in persistent activation of the molecule itself and the mutations have been called constitutively active form of Rac1 (ca Rac1). The ca Rac1 has been reported in human mammary carcinoma as a variant of the Rac1b, which harbors a 19 nt insertion in switch motif of Rac1. The mutation in Rac1 has been related to pathogenesis in human mammary cancers. Now we propose a hypothesis that the Rac1 variation may be a mutation of ca Rac1. It encodes a persistent activating protein, whereby to change the phynotypes of gastric epithelial cells including the aberrant activation for transcription, so to bring about the gastric carcinoma. It is worthy to make an investigation on the subject further. The novel points have been described one by one as above: summarily as follows:It is the 1st time to quantitate the micrometastasis of gastric carcinoma by a RTFQ-PCR and establish an integral experimental system to remark its accuracy, sensitivity, and specificity. It is the first time to develop the RTFQ-PCR analyzing method for Tiam1,a member of the network of cell signaling transduction. By its application, the clinical and pathlogical meanings for the quantification of Tiam1 transcripts was studied. It is the first time to raise the concept that the super-overexpression of both Tiam1 and Rac1 can be considered the definite hallmarker for the malignency-related aberrant activation for transcription. Its biological significance be the same as other experimental assays. It is the 1st time to reveal that gastric carcinoma-related over-expression of Tiam1 and Rac1 can be considered as a characterized signation for abnormal transcription activation responsible for pathogehesis of the illness. It is the first time to raise a hypothesis to link the super-overexpression of Tiam1 as well as Rac1 with carcinoma-related aberrant activation for transcription and the pathogenesis of gastric cancer. Because of the dependence of biological phynotypes on the genetic one, the aberrant transcription activation in malignant carcinomas take a most important part in the clinico-pathology and pathogenesis.
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