Increased activities of thymidine kinase isozymes in human colon polyp and carcinoma

Thymidine kinase (TK), the enzyme in the pyrimidine salvagepathway, and its isozymes were examined in 15 specimens of normalmucosa, 14 polyps and 14 carcinomas in human colon. The averageTK activities in colon polyps and carcinomas were about 1.6and 2.9 times that in normal colon mucosa, respectively. Thecolon TK isozymes were separated into 2 types by DEAE-cellulosecolumn chromatography. The activity of the TK isozyme elutedwith 0.1 M NaCI in buffer was 1.6-fold higher in colon polypand 1.5-fold higher in colon carcinoma than that in normal colonmucosa. In colon carcinoma, but not colon polyp, the activityof the other isozyme eluted with buffer alone was increasedto 5.1-fold that in normal tissues. As the activity of the latterisozyme was not affected by deoxycytidine triphosphate, it maybe involved in DNA replication. The results suggest that increasesin the activities in the former and latter TK isozymes may indicatetumorigenesis and carcinogenesis, respectively, in the humancolon.     

Increased activity of thymidine kinase isozyme in human colon tumor

Thymidine kinase (TK), the salvage pathway enzyme, and its isozymeswere examined in normal mucosa and carcinoma of the colon in13 patients. In colon tumor TK activity increased to 328% ofactivity of normal colon. The colon TK isozymes were separatedfrom the normal mucosa and carcinoma by DEAE-cellulose columnchromatography. The TK isozyme eluted from the column by theelution buffer alone without NaCl was markedly higher (12-fold)in activity in carcinoma than in normal mucosa of the colonand is not affected by dCTP. The TK isozyme unaffected by dCTPhas also been reported to be dearly involved in DNA synthesis.This isozyme, whose molecular weight is 100 000 by means ofhigh performance liquid chromatography, is thought to be involvedin DNA replication. The results indicate the applicability ofthe molecular correlation concept to carcinogenesis of colon.     

Thymidine kinase activities in sera and liver tissues during hepatocarcinogenesis in rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). In the present study, we investigated serum levels of alpha-fetoprotein (AFP) and thymidine kinase (TK), DNA-synthesizing enzyme in the salvage pathway, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly right after the onset of 3'-MeDAB treatment, peaking after one week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into 3 types by DEAE-cellulose column chromatography. A 3'-MeDAB induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that biochemical changes in 3'-MeDAB-treated rat liver may provide a valuable insight into two step process in hepatocarcinogenesis.     

Thymidine kinase and alpha-fetoprotein as biochemical markers of hepatocarcinogenesis induced by 3'-methyl-4-dimethylaminoazobenzene treatment in rats.

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by such azo dye carcinogens as 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). Thymidine kinase (TK) catalyzes the formation of deoxythymidine monophosphate (dTMP) by the phosphorylation of thymidine via the salvage pathway. In the present study, we investigated serum alpha-fetoprotein (AFP) and TK levels, and tissue TK and its isozyme activities in the liver of rats treated with 3'-MeDAB. Serum TK activities rose abruptly directly after the onset of 3'-MeDAB treatment, peaking after 1 week and then gradually decreasing. At 3 weeks, though serum TK was decreasing, serum AFP and tissue TK began to increase, and oval cells appeared in the liver. At 5 weeks, though serum TK reached a nadir, serum AFP and tissue TK formed transient peaks, and oval cells occupied a major part of the hepatic lobules with hyperplastic nodules. Thereafter, serum TK continued to increase, and serum AFP and tissue TK, after transiently decreasing, re-increased; at 20 weeks, each value was at high level, and mixed type hepatocarcinoma was observed. The liver TK isozymes were separated into three types by DEAE-cellulose column chromatography. A 3'-MeDAB diet induced a remarkable increase in activity of cytosolic and fetal type isozyme in non-tumorous regions of livers at 5 weeks and tumorous regions at 20 weeks. These results indicate that early biochemical changes in 3'-MeDAB-induced hepatocarcinoma in rats may serve as a useful model and provide a valuable insight in hepatocarcinogensis.     

Activity of the cytosolic isozyme of thymidine kinase in human primary lung tumors with reference to malignancy

Activity increase of the cytosolic isozyme of thymidine kinase (TK) in resected specimens of lung tumor patients would be a useful marker for tumor malignancy and prognosis. In 24 resected cases of malignant lung tumors, the whole enzyme extracts of the tumorous part of the specimens showed that the activities of TK, thymidylate synthetase, and ribonucleotide reductase increased at an average of 469 (P less than 0.001), 208 (not significant), and 193% (P less than 0.02) of the corresponding enzymes in the tumor-uninvolved lung parts, respectively. Two TK isozymes, cytosolic and mitochondrial TKs, were separated better by means of p-aminophenyl 3'-TMP:CH-Sepharose gel affinity column chromatography for precise quantitation of the activity than by polyacrylamide disc gel electrophoresis. These separated isozymes from the tumorous part of the specimens were characteristically very similar to the isozymes of cytosolic and mitochondrial fractions of the xenograft (CPX-101) of human lung tumor transplanted in athymic nude mice, respectively. The cytosolic isozyme activity isolated by this method from the tumorous part was remarkably higher and more varied than that of the tumor-uninvolved part, while that of the mitochondrial isozyme was lower and less agitated. The tumor doubling time showed a good inverse correlation to the activity of the cytosolic isozyme of TK when compared logarithmically (r = -0.798, P less than 0.01). Poorly differentiated tumors exhibited significantly higher activities of the TK cytosolic isozyme than did well-to-moderately differentiated tumors (766.0 +/- 379.1 and 308.1 +/- 119.5 pmol/mg of protein/h, mean +/- SE, respectively), a phenomenon also seen in the activities of the tumors with versus without recurrences within 12 mo after resection (803.6 +/- 278.7 and 124.1 +/- 42.1 pmol/mg of protein/h, respectively). The levels of these relationships using the cytosolic TK activity provided a clearer indication of prognosis and the state of the malignancy than those using the whole extract TK activity.     

Relative activities of thymidylate synthetase and thymidine kinase in 1,2-dimethyihydrazine-induced colon carcinomas in rats

Thymidylate synthetase (TS) and thymidine kinase (TK) are knownto catalyse the methylation of dUMP for the de novo synthesisof dTMP and the phosphorylation of thymidine for the salvagesynthesis of dTMP in the pyrimidine pathway, respectively. HighTS and TK activities and the existence of TK isozymes have beenobserved in rapidly proliferating tissues. TS and TK activitiesin 1,2-dimethyihydrazine (DMH)-induced colon carcinomas in ratsincreased significantly to 331 and 207% of the activities innormal colon, respectively, and were well correlated inversely(y = –0.93_x + 5.24), with a correlation coefficient of–0.787. The colonic TK iso zymes were separated into twotypes by DEAE-cellulose column chromatography. The TK isozymeeluted from the column by the elution buffer alone without NaClwas marked ly higher (23.6-fold) in activity in DMH-inducedcolon carci noma than in normal control colon and was not affectedby deoxycytidine triphosphate. This isozyme, whose mol. wt is100 000 by h.p.l.c., is thought to be closely involved in rapidDNA replication. These results indicate that early biochemicalchanges in DMH-induced colon carcinoma in rats may serve asa useful model and provide valuable insight into the mech-anismsinvolved in colonic carcinogenesis.     

Thermal effects on DNA synthesis in bone marrow cells of patients with acute myeloblastic leukemia.

It has been known that thymidylate synthetase (TS) and thymidine kinase (TK) are DNA-synthesizing enzymes via the de novo and salvage pathways, respectively, in pyrimidine metabolism, and an immunological method using a monoclonal antibody to bromodeoxyuridine (BrdU) is useful for the detection of S-phase cells. We investigated the effects of hyperthermia on DNA synthesis in bone marrow cells obtained from patients with acute myeloblastic leukemia. TK isozymes in bone marrow cells of patients with acute myeloblastic leukemia were separated into two types, i.e., fetal type isozyme in cytosolic cell fraction and adult type isozyme in mitochondrial cell fraction. Heating at over 43 degrees C as a hyperthermia markedly suppressed enzyme activity of fetal type TK isozyme and number of S-phase cells labelled with BrdU, but not activities of adult type TK isozyme and TS. These results indicate that hyperthermia may regulate the proliferation of S-phase cells by the suppression of salvage synthesis of DNA.     

大肠肿瘤APC基因突变的研究

目的检测ＡＰＣ基因在国人散发性大肠肿瘤中的突变情况,并探讨ＡＰＣ基因突变与大肠肿瘤生物学行为的关系。方法采用ＰＣＲＳＳＣＰ、ＤＮＡ测序对２７例（３３份标本）大肠息肉,３６例大肠癌和相应正常粘膜及３例家族性腺瘤性息肉瘤（ＦＡＰ）标本中ＡＰＣ基因“突变密集区”（ｍｕｔａｔｉｏｎｃｌｕｓｔｅｒｒｅｇｉｏｎＭＣＲ）的突变进行了研究。结果大肠息肉和癌组织中,ＡＰＣ基因ＭＣＲ突变率分别为３０．３％（１０／３３）和３５．９％（１４／３９）,两者差异无显著性,而正常大肠粘膜、炎性息肉和增生性息肉均无突变。３例ＦＡＰ发现有２例突变,其中１例从正常粘膜到腺瘤、到癌变组织均有突变,另１例则在＞１．０ｃｍ的腺瘤和肝转移癌组织有突变,测序结果证实该例在１４２５号密码子存在相同点突变。ＡＰＣ突变与肿瘤的临床病理特征无明显的相关性。结论ＡＰＣ不仅在ＦＡＰ中存在突变,而且在散发性大肠肿瘤中亦存在突变;它的突变至少参与了部分大肠癌的发生发展过程,而且是这一过程中较早发生的事件。     

Immunohistochemical localization of glutathione S-transferases alpha, mu, and pi in normal tissue and carcinomas from human colon.

Analysis of the heterogeneity of glutathione S-transferase (GST) isozyme expression was carried out by immunohistochemical evaluation of human colon biopsy tissue from 30 patients. Using polyclonal antibodies specific for the GST alpha, mu and pi families of isozymes, an increased expression of pi was found in 21/30 carcinoma specimens compared to their pair-matched controls. This isozyme was the most prevalent in all colon samples. GST mu was expressed at reduced levels in 20/30 carcinoma specimens when compared to normal. GST alpha showed no consistent change. Analysis of the immunostaining in different cell types showed that the highest intensity stain for all isozymes was in the columnar epithelial cells. These cells were primarily responsible for the proportional changes in GST pi and mu between carcinoma and normal tissues. In addition, goblet (crypt), endothelial and muscle cells stained positively. In the lamina propria, lymphocytes and phagocytes stained positively, while fibroblasts, plasma cells and leukocytes were negative. Endocrine cells were also negative. The differential expression of GST pi and mu, confirming biochemical data, supports the potential utility of GST pi as a carcinoma marker.     

Expression of a novel carbonic anhydrase,CA XIII,in normal and neoplastic colorectal mucosa

Background  

Carbonic anhydrase (CA) isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples.     

Expression of tumor rejection antigens in colorectal carcinomas

BACKGROUND: The authors recently reported that the SART2 and SART3 antigens encode tumor epitopes recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs) established from esophageal carcinoma patients. The current study investigated these antigens to explore a potential molecule for specific immunotherapy for colorectal carcinoma patients. METHODS: The SART2 and SART3 antigens were investigated by Western blotting in colorectal carcinoma cell lines and in cancer tissues. For induction of CTLs, peripheral blood mononuclear cells (PBMCs) of HLA A-24-positive cancer patients were stimulated in vitro with peptides. RESULTS: The 140 kD SART3 antigen was expressed in both the cytosol and nuclear fractions of all six colon carcinoma cell lines, 27 of 41 (65.9%) cytosol fractions, 30 of 41 (73.2%) nuclear fractions of colorectal carcinoma tissue samples, and in 0 of 7 non-tumorous tissues. The 100 kD SART2 antigen was expressed in the cytosol fractions of 2 of 6 colon carcinoma cell lines, 5 of 20 (25%) cytosol fractions of colorectal carcinoma tissue samples, and in 0 of 7 non tumorous tissues. HLA-A24-restricted CTLs cytotoxic to colon carcinoma cells were induced from PBMCs of colon carcinoma patients by stimulation with the two immunogenic peptides of SART3. CONCLUSIONS: The SART3 antigen could be an appropriate target molecule for specific immunotherapy for colorectal carcinoma patients.     

Significance of alkaline phosphatase isozymes in the monitoring of patients with colorectal carcinoma

The clinical course of colorectal carcinoma may be monitored by tumor markers such as carcinoembryonic antigen (CEA), carcinoma antigen (CA) 19-9 and CA-50. Alkaline phosphatase isozymes were previously used to study the clinical course of testicular and gynecologic tumors. In this study we investigated 8 patients with advanced colorectal carcinoma. Their sera were analyzed for the tumor markers CEA, CA 19-9, CA-50 and three alkaline phosphatase isozymes: the nonspecific liver isozyme LAP, the intestinal isozyme IAP and the placental isozyme PLAP. Rising levels of CEA, CA 19-9 and CA-50 were seen as expected, and PLAP also showed rising levels during tumor progression. LAP remained elevated. This indicates an association between progression of colorectal carcinoma and a raised serum content of alkaline phosphatase isozymes.     

Increased levels of promutagenic etheno-DNA adducts in colonic polyps of FAP patients

Nonsteroidal anti-inflammatory drugs (NSAIDs) can regress adenomas in patients with familial adenomatous polyposis (FAP), and the mechanism involves inhibition of cyclooxygenases (COX). Reactive intermediates formed during the arachidonic acid cascade, notably by COX-2, which is upregulated in polyps of FAP patients, may promote various stages of the polyp --> adenoma --> carcinoma sequence. Etheno-DNA adducts can be derived from reactive intermediates generated during arachidonic acid metabolism and lipid peroxidation. We tested this hypothesis in colonic polyps from FAP patients and colorectal tissue from cancer patients to see whether increased formation of etheno-DNA adducts occurs. Using an ultra-sensitive and specific immunoaffinity/(32)P-postlabelling method, 1, N(6)-ethenodeoxyadenosine (straightepsilondA) and 3, N(4)-ethenodeoxycytidine (straightepsilondC) were quantitated in epithelial cell DNA from asymptomatic colon, FAP polyps and colon tumor tissues. Mean adduct levels in FAP polyps were 65 straightepsilondA/10(9) and 59 straightepsilondC/10(9) parent nucleotides, being 2 to 3 times higher than in unaffected colon tissue (p < 0.02 for straightepsilondA; p < 0.05 for straightepsilondC). Adduct levels in colonic epithelia decreased in the order: FAP polyps > tumor-adjacent tissue > tumor, normal and tumor-distal tissue. Based on this study, requiring confirmation in a larger number of patients and in experimental models, we have demonstrated the formation of promutagenic etheno-DNA adducts in adenomatous polyps of FAP patients that may contribute to genetic instability and cancer progression.     

beta-Hexosaminidase from colon and sera of dukes-classified colorectal cancer patients: activity levels, isozyme patterns, and kinetic properties

Total activity levels, isozyme patterns, and kinetic properties of beta-hexosaminidase were studied in crude supernatants of malignant and adjacent, uninvolved normal colon tissues and in sera from 28 Dukes-classified colorectal cancer patients. A significant increase (P less than .001) in both beta-hexosaminidase activity and beta-hexosaminidase specific activity and a significant increase (P less than .01) in the relative percentage of activity comprised by the basic thermostable form of beta-hexosaminidase (Hex B) isozyme were found in malignant tissue compared to the activities seen in uninvolved normal colon tissue. No apparent correlations were found between either beta-hexosaminidase activity levels or relative percentage of Hex B and Dukes category. Kinetic analysis indicated that the thermolabile form of beta-hexosaminidase and Hex B from malignant colon were comparable to the corresponding isozymes from normal colon with regard to thermostability after preincubation at 50 degrees C and pH activity curves (optimum between pH 4.0 and 5.0). Significantly decreased (P less than .05) beta-hexosaminidase activity was found in sera of the 28 colorectal cancer patients (17.3 +/- 5.2 U/ml, mean +/- SD) when compared to the activity in 19 controls with nonmalignant diseases (21.4 +/- 8.2 U/ml) and 17 normal controls (21.3 +/- 6.4 U/ml). Isoelectric focusing indicated that a peak of beta-hexosaminidase activity with an isoelectric point value (9.5) comparable to that of the peak found in increased amounts in malignant colon was detectable in the sera of 36% of the colorectal cancer patients and 11% of the controls.     

Effects of neo-adjuvant chemotherapy with UFT (a combination of tegafur and uracil) on DNA-synthesizing enzyme activities in human colorectal carcinomas

Thymidylate synthetase (TS) and thymidine kinase (TK) are known to catalyse the methylation of dUMP for the de novo synthesis of dTMP and the phosphorylation of thymidine for the salvage synthesis of dTMP in the pyrimidine pathway, respectively. High TS and TK activities have been observed in rapidly proliferating tissues such as foetal tissue, regenerating liver and carcinomas. TS and TK activities were measured in histologically normal colon mucosa and colorectal carcinomas with or without neo-adjuvant chemotherapy of an antitumor drug UFT (a combination of tegafur and uracil) in humans. In colorectal carcinomas, 5-FU and uracil levels were increased to 2.6- and 3.2-fold, respectively, those in normal colon mucosa by neo-adjuvant chemotherapy of UFT. In colorectal carcinomas, TS and TK activities were both increased to approximately 2- and 3-fold, respectively, those in normal colon mucosa without UFT treatment. In normal colon mucosa and colorectal carcinomas with neo-adjuvant chemotherapy of UFT, TS activities were reduced to approximately 50% and 40%, respectively, those without UFT treatment. These results indicate that neo-adjuvant chemotherapy with UFT may prevent dissemination of cancer cells during operation, and local recurrence and distant metastasis after operation, inhibiting DNA synthesis via the de novo pathway of pyrimidine synthesis in carcinomas.     

A randomized placebo-controlled prevention trial of aspirin and/or resistant starch in young people with familial adenomatous polyposis

Evidence supporting aspirin and resistant starch (RS) for colorectal cancer prevention comes from epidemiologic and laboratory studies (aspirin and RS) and randomized controlled clinical trials (aspirin). Familial adenomatous polyposis (FAP) strikes young people and, untreated, confers virtually a 100% risk of colorectal cancer and early death. We conducted an international, multicenter, randomized, placebo-controlled trial of aspirin (600 mg/d) and/or RS (30 g/d) for from 1 to 12 years to prevent disease progression in FAP patients from 10 to 21 years of age. In a 2 × 2 factorial design, patients were randomly assigned to the following four study arms: aspirin plus RS placebo; RS plus aspirin placebo; aspirin plus RS; RS placebo plus aspirin placebo; they were followed with standard annual clinical examinations including endoscopy. The primary endpoint was polyp number in the rectum and sigmoid colon (at the end of intervention), and the major secondary endpoint was size of the largest polyp. A total of 206 randomized FAP patients commenced intervention, of whom 133 had at least one follow-up endoscopy and were therefore included in the primary analysis. Neither intervention significantly reduced polyp count in the rectum and sigmoid colon: aspirin relative risk = 0.77 (95% CI, 0.54-1.10; versus nonaspirin arms); RS relative risk = 1.05 (95% CI, 0.73-1.49; versus non-RS arms). There was a trend toward a smaller size of largest polyp in patients treated with aspirin versus nonaspirin--mean 3.8 mm versus 5.5 mm for patients treated 1 or more years (adjusted P = 0.09) and mean 3.0 mm versus 6.0 mm for patients treated more than 1 year (P = 0.02); there were similar weaker trends with RS versus non-RS. Exploratory translational endpoints included crypt length (which was significantly shorter in normal-appearing mucosa in the RS group over time) and laboratory measures of proliferation (including Ki67). This clinical trial is the largest ever conducted in the setting of FAP and found a trend of reduced polyp load (number and size) with 600 mg of aspirin daily. RS had no clinical effect on adenomas.     

Mutations of the PIK3CA gene in hereditary colorectal cancers

Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients.     

Telomerase activity in colorectal carcinoma and its correlation with expression of c-myc

Objective： To study the role of telomerase activity and c-myc in pathogenesis and progression of colorectal carcinoma, and to investigate the possible regulatory mechanism of telomerase activation. Methods： A modified telomeric repeat amplification protocol （TRAP） and immunohistochemical staining was used to detect telomerase activity and the expression of c-myc in tissue samples from colorectal carcinoma, paracarcinomatousl tissues, normal mucosa, and adenomatoid polyp. Results： The positive rates of telomerase activity and c-myc expression were 83.33% and 80.00% in colorectal carcinoma, 13.33% and 23.33% in paracarcinomatousl tissues, 13.33% and 20.00% in normal mucosa, and 10.00% and 45.00% in adenomatoid polyp respectively, they were significantly higher in colorectal carcinoma than in paracarcinomatousl tissues, normal mucosa, and adenomatoid polyp （P〈0.05）. The rates of telomerase activity and c-myc expression were much higher in colorectal carcinoma with lymph nodes metastases than that without lymph nodes metastases. The expression of c-myc was found being significantly higher in the telomerase positive colorectal carcinoma than in the telomerase negative group （P〈0.05）. Conclusion： The activation of telornerase and abnormal expression of c-myc might play an important role in the process of carcinogenesis and progression of colorectal carcinoma. The over-expression of c-myc may be related to telomerase activation and up-regulation in colorectal carcinoma.