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Biomedical subjects

G J Peters

Publications and source records attributed to G J Peters.

At least 91 records · Page 5Linked to original sources
PubMed2001

Rb, mcl-1 and p53 expression correlate with clinical outcome in patients with liver metastases from colorectal cancer.

BACKGROUND: Thymidylate synthase (TS) has been associated with clinical outcome in disseminated colorectal cancer. However, many patients with low TS expression still fail to respond to treatment. Therefore, we studied the cell cycle proteins, Rb, E2F2, Ki67, p21 and p53 and the apoptotic proteins, mcl-1, hax, bcl-xl, bcl-2, Fas receptor, Fas ligand, caspase-3, M30 and PARP as potential predictive factors. PATIENTS AND METHODS: In biopsy specimens of liver metastases from 31 colorectal cancer patients, protein expression was retrospectively determined by immunohistochemistry and related to response to hepatic arterial or intravenous (i.v.) 5-fluorouracil (5-FU) treatment, time to tumour progression (TTP) and overall survival. RESULTS: Expression of both p53 and Rb correlated with survival benefit after 5-FU treatment. A median survival time of 79 weeks was found in patients with high levels of p53 or Rb compared to 36 and 44 weeks for patients expressing low levels of p53 (P = 0.027) or Rb (P = 0.030), respectively. Multivariate analysis showed that p53 was the best predictor of survival independent of sex, age or prior treatment. Following 5-FU hepatic arterial infusion, patients with a high TS expression had a shorter survival time than those with a low expression (P = 0.025). The anti-apoptotic protein mcl-1 was the only factor, which correlated with response to 5-FU treatment. Thirty-five percent of patients with a diffuse mcl-1 expression responded whereas ninety percent of patients with a peri-nuclear expression responded (P = 0.041). CONCLUSIONS: These results indicate that besides TS, also Rb, p53 and mcl-1 are correlated with clinical outcome in patients with liver metastases from colorectal cancer.

Adult↗
PubMed2001

Issues of normal tissue toxicity in patient and animal studies--effect of carbogen breathing in rats after 5-fluorouracil treatment.

Non-invasive magnetic resonance spectroscopy (MRS) can be used in the clinic to monitor the pharmacokinetics of the chemotherapeutic drug 5-fluorouracil (5-FU) and the effects of modifiers. We report two studies of 5-FU toxicity in normal tissue--one with patients and the other an animal study. 1) 19F MRS signals from fluoronucleotides, cytotoxic anabolites of 5-FU metabolism, were observed in the livers of two patients treated with 5-FU for colorectal cancer, shown by computed tomography (CT) and ultrasound (US) to have no liver metastases. This is the first report of non-invasive monitoring of toxic 5-FU metabolites in normal human tissues. 2) In animals, carbogen-breathing enhances tumour uptake and the efficacy of 5-FU, and the method is under trial in patients. This study demonstrates that there were no significant effects of carbogen breathing on the levels of 5-FU and its metabolites in normal rat tissues, or on the histology of the tissues assessed after treatment.

Adenocarcinoma↗
PubMed2001

Irinotecan and oxaliplatin: an overview of the novel chemotherapeutic options for the treatment of advanced colorectal cancer.

Colorectal cancer is one of the most frequent malignancies in humans and an important cause of cancer death. Metastatic colorectal cancer remains incurable with available systemic therapeutic options. The most active cytotoxic drug against this malignancy, the antimetabolite 5-fluorouracil, was developed more than forty years ago, and as a single agent produces responses in only 10 to 15% of patients which in general last less than one year. Efforts to ameliorate these poor results resulted in the 5-fluorouracil/leucovorin combination, which enhances response rates about two-fold, without, however, significantly improving survival rates. The recent emergence of a handful of new 5-fluorouracil analogues and folate antagonists, as well as the topoisomerase I inhibitor irinotecan, and the third-generation platinum compound oxaliplatin, is likely to alter this gloomy scenario. These agents are at least as effective as 5-fluorouracil in patients with advanced colorectal carcinoma, both untreated and previously treated with 5-fluorouracil-based regimens. This has led to the approval of irinotecan as second-line treatment for 5-fluorouracil-refractory disease, while the use of oxaliplatin has been suggested for patients having a defective 5-fluorouracil catabolism. Recently, FDA approved the combination of irinotecan with 5-fluorouracil and leucovorin for first-line treatment of advanced colon cancer. Based on the synergistic preclinical antitumor effects of some of these agents, their meaningful single-agent activity, distinct mechanisms of cytotoxicity and resistance, and only partially overlapping toxicity profiles, effective combination regimens are now being developed, which are likely to lead to a new, more hopeful era for patients suffering from advanced colorectal carcinoma.

Antimetabolites, Antineoplastic↗
PubMed2001

Chemotherapeutic strategies for treatment of colorectal cancer: present and future developments.

Colorectal cancer is one of the most common malignancies in Western nations. Treatment with 5-fluorouracil and leucovorin resulting in inhibition of DNA synthesis after inhibition of thymidylate synthase is the most common strategy for adjuvant and systemic chemotherapy in this disease. However, the majority of patients still fail to respond to this combination; therefore, new chemotherapeutic strategies have been developed. This process has proceeded rapidly due to a more proper translation of preclinical results to the clinic. Knowledge of proximal parameters (drug activation, drug-target interaction) and distal parameters (cell growth inhibitory parameters and cell death parameters) in drug response should offer the opportunity to discriminate between sensitivity and resistance to chemotherapy and therapeutic selectivity. This clinical commentary presents a review of a symposium held at the Vrije Universiteit Medical Center, Amsterdam, The Netherlands, which focused on both the preclinical and clinical aspects relating to the treatment of colorectal cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Interference of gemcitabine triphosphate with the measurements of deoxynucleotides using an optimized DNA polymerase elongation assay.

The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.

Antimetabolites, Antineoplastic↗
PubMed2000

Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460.

Chemotherapy-induced apoptosis is generally thought to be dependent on a pathway headed by caspase-9. This model is primarily based on studies performed in leukemia cells; however, little is known about caspase cascades in relatively resistant solid tumor cells, including non-small cell lung cancer (NSCLC) cells. Using the NSCLC cell line NCI-H460 (H460), here, we studied the effect of stable expression of various caspase inhibitors on apoptosis induced by the anticancer drugs cisplatin, topotecan, and gemcitabine. Interestingly, overexpression of caspase-9S and X-linked inhibitor of apoptosis (XIAP), both able to inhibit caspase-9 activity, failed to block apoptosis. In contrast, stable expression of caspase-8 inhibitors, such as cytokine response modifier A (CrmA) and dominant-negative caspase-8, almost completely abrogated apoptosis and also enhanced clonogenic survival. Caspase-8 activation in H460 cells was not mediated by death receptors, inasmuch as overexpression of dominant-negative Fas-associated death domain (FADD-DN) did not prevent procaspase-8 cleavage and subsequent apoptosis. However, stable expression of Bcl-2 and Bcl-xL did suppress these apoptotic events, including the release of cytochrome c from mitochondria, which was observed in drug-treated H460 cells. In the NSCLC cell line H460, we, thus, provide evidence for the existence of a novel drug-inducible apoptotic pathway in which activation of caspase-8, and not of caspase-9, forms the apical and mitochondria-dependent step that subsequently activates the downstream caspases.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells.

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258/propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator/urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFalpha or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.

Antineoplastic Agents↗
PubMed2000

Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake.

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.

Antimetabolites, Antineoplastic↗
PubMed2000

Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates.

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.

Animals↗
PubMed2000

L-Dopa stimulates expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO) in cultured astroglial cells.

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.

Animals↗
PubMed2000

Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines.

To gain a more detailed insight into the metabolism of 2', 2'-difluoro-2'-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R=-0.61), but also the intrinsic CTP/UTP ratio (R=0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6-1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R=0.64 (P<0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC.

Animals↗
PubMed2000

Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed.

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs.

Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Schedule-dependent pharmacodynamic effects of gemcitabine and cisplatin in mice bearing Lewis lung murine non-small cell lung tumours.

The combination of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) is increasingly applied in clinical oncology. We studied the underlying mechanisms of the in vivo schedule dependency and supraadditive interaction between dFdC and CDDP in C57/B16 mice bearing Lewis lung (LL) tumours. Mice were treated with CDDP (6 mg/kg) and dFdC (60 mg/kg) either simultaneously or in a 4 or 24 h interval with dFdC preceding CDDP or vice versa. Four, 8 (in some cases 12) and 24 h after treatment mice were sacrificed and tumours, kidneys, blood and bone marrow (BM) were collected. Since CDDP acts by formation of Platinum (Pt)-DNA adducts and dFdC by incorporation of its triphosphate (dFdCTP) into DNA, we measured total Pt levels, dFdCTP accumulation and Pt-DNA adducts by atomic absorption spectrometry (AAS), high performance liquid chromatography (HPLC) and 2P-postlabelling, respectively. These levels were related to the previously determined antitumour efficacy and toxicity of the dFdC/CDDP combination. Peak dFdCTP accumulation in tumours (11 pmol/mg) was found 4 h after dFdC treatment, while CDDP tended to reduce this in a time-dependent way. Peak levels of total Pt in tumours were found 4 h after CDDP treatment (581 fmol/mg) and dropped 1.8-fold after simultaneous treatment with dFdC (P = 0.04). Treatment with dFdC 4 h after or simultaneously with CDDP increased Pt retention (level 24 h after CDDP treatment) 1.4- and 1.6-fold (P = 0.04 and P = 0.03, respectively). Peak Pt-DNA adduct levels in tumours were also found 4 h after CDDP treatment (7 fmol/microg DNA) and were decreased 3-fold by dFdC treatment 24 h prior to CDDP (P = 0.04). Pt-DNA adduct retention was only decreased when dFdC was given 4 h before CDDP (8-fold (P < 0.01)). The retention and the area-under the concentration time curve of Pt-DNA adducts were related to decreased tumour doubling time (linear regression coefficient (R) = 0.95; P < 0.05, 0.96 P = 0.04 and 0.90; P = 0.04. Pt-DNA adduct levels in the BM cells reached a plateau level 4-24 h after CDDP treatment (approximately 10 fmol/microg DNA), which was increased by dFdC when given either simultaneously with, 4 h before or 4 h after CDDP (6-, 3- and 5-fold at 28 h, 8 h and 28 h, respectively (P < or = 0.04)). Peak Pt-DNA adduct formation (24 h: 8 fmol/microg DNA) in kidneys was enhanced by dFdC when given simultaneously with or 4 h before CDDP (4 h timepoint) (P < 0.01). However, retention was 4- and 6-fold decreased when dFdC was given 4 or 24 h after CDDP, respectively (P < or = 0.01). dFdC given 24 h before CDDP decreased all Pt-DNA adduct levels in kidneys 3-fold or more (P < or = 0.03). Pt-DNA adduct levels were inversely related to kidney toxicity when the most toxic schedule was excluded from the analysis. Peak levels of total Pt in kidneys were reached 24 h after CDDP treatment (4.3 fmol/mg) and the 8 h levels were increased 2-fold by dFdC when given 4 h after CDDP (P = 0.07).

Animals↗
PubMed2000

Downstream molecular determinants of response to 5-fluorouracil and antifolate thymidylate synthase inhibitors.

Thymidylate synthase (TS) is an essential enzyme for the de novo synthesis of thymidylate and subsequently DNA synthesis. TS has been used as a target for cancer chemotherapy in the development of fluoropyrimidines such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine and of novel folate-based TS inhibitors such as ZD1694 (Tomudex, Raltitrexed), ZD9331, LY231514 (ALIMTA, Pemetrexed), AG337 (Thymitaq, Nolatrexed) and AG331. Although TS has been considered as a target for chemotherapy, the precise mechanism by which TS inhibition leads to cell death is still not completely resolved. TS inhibition results in depletion of dTTP, an essential precursor for DNA, and an increase in dUTP. This results in the so-called thymine-less death due to misincorporation of dUTP into DNA; its excision, catalysed by uracil-DNA glycosylase, results in DNA damage. Both this imbalance in dTTP/dUTP and DNA damage can result in induction of downstream events, leading to apoptosis. On the other hand a specific interaction exists between oncogenes and TS, by binding of TS protein to the p53 and c-myc RNA, while wt p53 can also inhibit TS promotor activity. TS inhibition by either 5-FU or antifolates can also result in a depression of TS protein mediated inhibition of TS mRNA translation leading to induction of more TS protein synthesis, and p53 protein may further deregulate this process. These complex indirect and direct interactions between oncogenes and TS may have as yet unclear clinical implications, since most data are based on in vitro or in vivo studies and some results are contradictive. In some preliminary clinical studies evidence was postulated for a combined prognostic role for TS and p53. This knowledge should be used to design clinical studies with the aim to deliver effective treatment to potentially sensitive patients both in the adjuvant setting and in advanced stage disease.

Antimetabolites, Antineoplastic↗
PubMed2000

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Antimetabolites, Antineoplastic↗