H. H. J. Backus

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.

The multilayered postconfluent cell culture as a model for drug screening

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.

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients

BACKGROUND In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.

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 μM 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. Int. J. Cancer 87:771–778, 2000.

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.

Differences in the induction of DNA damage, cell cycle arrest, and cell death by 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is an important target for chemotherapy and can be inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can be incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will influence activation of downstream mechanisms mediated via TS inhibition. Therefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG337, and MTA to determine the induction of DNA damage, cell cycle arrest, downstream protein expression, and cell death. At these concentrations, the specific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo and WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). Western blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treated Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nevertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells differences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS inhibition.

Thymidylate Synthase Expression in Patients with Colorectal Carcinoma Using a Polyclonal Thymidylate Synthase Antibody in Comparison to the TS 106 Monoclonal Antibody

Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidin-biotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5,10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.

Thymidylate Synthase Inhibition Induces P53 Dependent and Independent Cell Death

Thymidylate synthase (TS) is an important target for chemotherapy in colon cancer. It is the rate limiting de novo enzyme for synthesis of thymine nucleotides, one of the precursors for DNA synthesis. TS can be inhibited by several clinically active compounds 1,2 such as 5-fluorouracil (5-FU) and the antifolates (AG337 (Thymitaq, Nolatrexed), ZD1694 (Tomudex, Raltitrexed), LY231514 (MTA or pemetrexed). This will lead to inhibition of DNA synthesis and imbalance in dUTP/dTTP pools which will result in DNA damage. DNA damage can trigger downstream events such as p533, bax 4, Fas receptor and caspase-3 to initiate apoptosis 5. It is however not completely known which mechanisms are involved in the onset of apoptosis after TS inhibition. Therefore, we studied the induction of downstream events after exposure of colon cancer cell lines to 5-FU and antifolates and the role that p53 plays during the process of apoptosis initiation.

5-Fluorouracil Induction of Fas and Apoptosis in Colon Cancer Patients: Relation of Clinical Outcome with Thymidylate Synthase, Mcl-1 and Rb

Thymidylate synthase (TS), catalyses the methylation of dUMP to dTMP with 5,10-methylene-tetrahydrofolate (CH2-THF) (1, 2). The active metabolite of 5-fluorouracil (5-FU), FdUMP forms a ternary complex with TS and CH2-THF leading to TS inhibition, growth arrest and cell death (1), which is stimulated by leucovorin (LV) (3, 4). Resistance to 5-FU is not only related to high TS levels, but also to mechanisms involved in cell growth inhibition or programmed cell death (3, 4, 5). Cell cycle progression is controlled by p53 (6), p21 (7), Rb and E2F (8). Drug treatment induces DNA damage, and p53 activates p21 to initiate cell cycle arrest. The tumor suppressor gene Rb forms a complex with E2F inhibiting their transcriptional activity (8). After phosphorylation of Rb by specific kinases, cell cycle progression can be activated by release of E2F1 and E2F2.

Induction of Resistance to the Multi-Targeted Antifolate MTA (LY231514) in Widr Human Colon Cancer Cells

The enzyme thymidylate synthase (TS) catalyses the methylation of 2′-deoxyuridine-5′monophosphate (dUMP) to 2′-deoxythymidine-5′-monophosphate (dTMP), an essential precursor during DNA synthesis (1). 5,10-Methylene tetrahydrofolate (CH2-THF) is the limiting methyl donor during this reaction. TS, is usually elevated in tumors (2) and is therefore an interesting target for anticancer agents such as the antifolate multi-targeted antifolate (MTA, ALIMTA, PEMETREXED)(3,4), which inhibits activity of TS by competition with the binding site of CH2-THF of TS. MTA is currently been developed as an anticancer agent for treatment of colorectal, non-small-cell lung cancer and mesothelioma (4). Resistance to this agent might develop similarly to other antifolates, and might be due to elevated activity or mutations of the target enzyme TS, impaired polyglutamation of antifolates, decreased transport into the cells, but also apoptosis regulating proteins may be involved (5,6). For this purpose we induced resistance to the antifolate MTA in the colon cancer cell line WiDr by exposure to 50 mM MTA for 4 hours every week, resulting in the WiDr-4LY cell line, and by continuous exposure for 72 hr to 20 mM MTA every week, resulting in WiDr-cLY. The resistant WiDr variants were studied on the level of TS and folyl polyglutamate synthetase (FPGS).