Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent cell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers. The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to observe cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools. Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase. P53 expression increased. Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers. Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g. the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions. Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting application is the possibility to perform drug combination studies. The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line). Neither of the drugs was independently able to produce similar effects. In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening.
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PMID:The multilayered postconfluent cell culture as a model for drug screening. 1103 3

We evaluated the efficacy of chemoradiotherapy (CRT) for advanced esophageal cancer, from the view point of response. The relationship between chemo-radiosensitivity and dihydropyridine dehydrogenase (DPD), thymidylate synthase (TS), and p53 was investigated immunohistochemically. Thirteen patients with inoperable advanced esophageal cancer were involved in this study. CDDP of 10 mg/m2/day and 5-FU of 335 mg/m2/day were infused intravenously (day 1-5, day 15-19). Radiation was delivered concomitantly at a total dose of 30 Gy. Expressions of p53, DPD and TS were detected using immunohistology in the biopsy samples taken before CRT from 8 patients. Partial response was observed in 8 cases, no change in 4 cases, and progressive disease in one case. The overall response rate was 62%. The reduction rate was higher in tumors positive for p53 expression than in negative ones. The same was true for DPD and TS. The Treatment effect was more precisely predicted by combination of p53, DPD and TS. CRT with low-dose CDDP + 5-FU chemotherapy was effective and combination with p53, DPD, and TS might be a predictive marker for CRT in patients with advanced esophageal cancer.
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PMID:[Chemoradiotherapy with low-dose cisplatin and 5-FU for advanced esophageal cancer]. 1108 19

The thymidylate synthase inhibitor raltitrexed (ZD1694, Tomudex) induces greater intestinal toxicity, manifested as diarrhea and weight loss, in BALB/c than in DBA/2 mice. No convincing pharmacokinetic or pharmacodynamic reason for this strain difference has been established. We have investigated whether this strain difference in response to raltitrexed is related to differential susceptibilities of intestinal mucosae to undergo apoptosis and also whether p53 expression, a critical factor in 5-fluorouracil-induced intestinal apoptosis and toxicity, modulates this response. Ten mg/kg or 100 mg/kg raltitrexed were administered as single or double i.p. injections 24 h apart to BALB/c, DBA/2, and p53-/- mice. Apoptosis, mitosis, and tissue damage were assessed in intestinal epithelium, and animal weight was recorded. BALB/c mice developed diarrhea and weight loss following 100 mg/kg x2 raltitrexed, whereas DBA/2 mice did not. BALB/c mice were more sensitive than DBA/2 to induction of small-intestinal and colonic apoptosis 24 h following 100 mg/kg raltitrexed. Inhibition of mitosis was equivalent in both strains. Both strains showed histopathological damage to the small intestine after 100 mg/kg x2 raltitrexed, but only BALB/c mice demonstrated colonic damage. p53-null mice showed the same level of small intestinal apoptosis as their wild-type counterparts 24 h after 100 mg/kg x1 raltitrexed and also the same levels of intestinal toxicity 3, 5, and 7 days after 100 mg/kg x2 raltitrexed. Thus, BALB/c mice were more susceptible to induction of intestinal apoptosis by raltitrexed than DBA/2 mice and also demonstrated more histopathological damage in the colon correlating with the induction of diarrhea and weight loss. In contrast to 5-fluorouracil, the intestinal apoptosis and toxicity induced by raltitrexed were p53-independent.
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PMID:The importance of p53-independent apoptosis in the intestinal toxicity induced by raltitrexed (ZD1694, Tomudex): genetic differences between BALB/c and DBA/2 mice. 1110 58

Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95-CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95-CD95L interactions.
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PMID:Apoptosis of haematopoietic cells upon thymidylate synthase inhibition is independent of p53 accumulation and CD95-CD95 ligand interaction. 1111 3

In this study, the downstream effects of thymidylate synthase (TS) inhibition in L1210 (p53 mutant) and HL60 (p53 null) leukaemia cells were investigated. TS inhibition was induced by the specific TS inhibitor Thymitaq. Within 24 h, TS inhibition resulted in S-phase cell cycle arrest in both cell lines and subsequent apoptotic cell death as characterized by nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. A biphasic hyper/hypopolarization of the mitochondrial membrane potential (delta psi m) was also observed. The mitochondrial permeability transition inhibitor, cyclosporin A, increased the baseline level of delta psi m in L1210 cells. However, along with bongkrekic acid, it did not influence the changes in delta psi m induced by TS inhibition in either cell line. In both cell lines the broad spectrum caspase inhibitor, zVAD.fmk as a single agent, induced a significant downward shift in the baseline of delta psi m. However, only in HL60 cells was this accompanied by a slight increase in cytotoxicity. In L1210 cells zVAD.fmk inhibited DNA fragmentation induced by Thymitaq but did not influence other cell cycle events (S-phase arrest) or the biphasic mitochondrial alterations, indicating caspase involvement downstream but not upstream of the mitochondria following TS inhibition. In HL60 cells, zVAD.fmk reduced the hyperpolarization of delta psi m observed with Thymitaq alone and failed to inhibit the increase in the sub-G1 population induced by Thymitaq. Moreover, zVAD.fmk significantly increased the cell death response of these cells following TS inhibition. In conclusion, cell death induced by TS inhibition is mediated via the apoptotic pathway which clearly involves biphasic alterations in delta psi m. In L1210 cells, but not in HL60 cells, caspases function as the final executioner of apoptosis.
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PMID:Thymidylate synthase inhibition induces S-phase arrest, biphasic mitochondrial alterations and caspase-dependent apoptosis in leukaemia cells. 1113 61

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2'-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5' upstream cis-acting regulatory element (nucleotides 80-109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of beta-actin, alpha-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.
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PMID:Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells. 1113 11

Preoperative chemotherapy and radiation (chemoradiation) are increasingly used in the treatment of advanced rectal carcinoma to downstage the tumor so that a sphincter sparing procedure is used. This treatment modality has also resulted in not only local disease control but also decreased metastasis and increased survival. It is well known that with standard chemoradiation some tumors show marked pathologic response, while others remain non-responsive. Identification of tumor markers that can predict responsiveness to chemoradiation is extremely useful to avoid unnecessary preoperative treatment. To understand the role of thymidylate synthase (TS), p53 and Bcl-2 proteins, if any, in tumor response/resistance to chemoradiation, we examined pretreatment biopsy material obtained from 12 responsive and 13 non-responsive patients by immunohistochemistry. TS was undetectable in 11 of 12 (92%) responsive tumors and overexpressed in only 1 tumor (8%); whereas, p53 or Bcl-2 was overexpressed in 8 tumors (66%). In the non-responsive group of 13 tumors, overexpression of TS, p53 and Bcl-2 was observed in 7, 5 and 6 tumors, respectively. In 6 non-responsive tumors in which TS was undetectable, 5 tumors contained high levels of p53 or Bcl-2. These results indicate that level of TS in tumors is the best predictor of sensitivity or resistance to chemoradiation. No such correlation between overexpression of p53 and Bcl-2 and response to chemoradiation is observed.
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PMID:Molecular markers and prediction of response to chemoradiation in rectal cancer. 1129 69

Gastrointestinal cancers account for a large amount of human tumors. Surgery is the standard treatment for localized gastrointestinal cancer, but in a large number of patients, tumors are unresectable at time of diagnosis and even when resectable, survival is often poor. Current attempts to improve these results include the use of chemotherapy in the adjuvant setting, in the advanced disease, or as neoadjuvant treatment. However, less than half the patients respond to chemotherapeutic treatments, mostly reporting important side-effects. The identification of molecular markers, such as p53, thymidylate synthase, K-ras, and others, may provide an important tool for medical oncologists in defining subsets of patients with gastrointestinal cancers more suitable to benefit from chemotherapy or from experimental therapies. The relationship between the clinical outcome to anticancer drugs and molecular markers in gastrointestinal tumors has been reviewed. Available data are promising, but most of them arise from retrospective and small studies. Well designed, prospective trials are warranted to change the target approach from a general to an individual treatment strategy.
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PMID:Molecular markers predictive of response to chemotherapy in gastrointestinal tumors. 1131 57

We developed a cell system where expression of thymidylate synthase (TS), an enzyme essential for DNA synthesis, can be modulated by a Zn(2+)-inducible promoter in MCF-7 cells. We found that overexpression of TS resulted in downregulation of p21 protein and mRNA levels. Statistical analysis demonstrated a significant downregulation of p21, but not a statistically significant decrease in p53 protein levels following TS induction. Since p21 is known to be transcriptionally activated by p53, these results suggest that TS downregulation of p21 may be occurring through a p53-independent mechanism in this in vitro cell system. In addition, cell cycle analysis demonstrated that downregulation of p21 by TS resulted in a decreased G(1)/S ratio in MCF-7 cells.
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PMID:Downregulation of p21/WAF1 expression by thymidylate synthase. 1144 25

We studied apoptosis and thymidylate synthase (TS) inductions by 5-fluorouracil (5-FU) in gastric cancer cells with wild-type p53 (MKN-45 and MKN-74) and with mutated p53 (MKN-28 and KATO-III). Apoptotic inductions in MKN-45 and MKN-74 were stronger than those in MKN-28 and KATO-III, suggesting that wild-type p53 may contribute to the induction of apoptosis. After continuous exposure to 0.1 microg/ml of 5-FU for 96 h, no TS induction was obtained in KATO-III, while approximately twice the amount of TS was observed compared to non-treatment cells in MKN-45, MKN-74, and MKN-28. The results of immunohistochemical staining for TS and p53 showed no relation between these two protein expressions in endoscopic biopsy specimens of 25 patients with advanced gastric cancer. These results indicated that p53 status may not play a pivotal role in regulating TS expression. We found no significantly different effects of 5-FU between intermittent (repeat of 24-h continuous infusion and 24-h drug-free) and continuous treatments in either MKN-28 or stem cells (CD34+ hematopoietic progenitor cells) when the same area under the time-concentration curve of 5-FU was administered. The TS induction in MKN-28 cells by intermittent treatment was significantly higher than that by continuous treatment; however, most TSs in both types of 5-FU treatment cells were of the inactive form, i.e., TS bound to FdUMP, a 5-FU metabolite. Therefore, neither intermittent nor continuous treatment appears to induce 5-FU resistance related to the level of increment free TS. In conclusion, our observations suggested that p53 mutation may be associated with apoptotic induction by 5-FU; however, p53 status may not strongly affect TS induction by 5-FU. Intermittent treatment can be replaced with continuous treatment without causing 5-FU resistance.
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PMID:Apoptosis and thymidylate synthase inductions by 5-fluorouracil in gastric cancer cells with or without p53 mutation. 1144 54


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