Biomedical subjects

G J Peters

Publications and source records attributed to G J Peters.

At least 163 records · Page 9Linked to original sources

PubMed1997

The antiproliferative effect of 8-chloro-adenosine, an active metabolite of 8-chloro-cyclic adenosine monophosphate, and disturbances in nucleic acid synthesis and cell cycle kinetics.

8-Chloro-adenosine, the dephosphorylated metabolite of the antineoplastic agent 8-chloro-cyclic AMP, has been proposed to act on the regulatory subunits of cyclic AMP-dependent protein kinase. 8-Chloro-adenosine has a growth-inhibitory effect, the mechanism of which is unclear. We investigated the effects of 8-chloro-cyclic AMP and 8-chloro-adenosine on nucleic acid synthesis and cell cycle kinetics in two human glioma cell lines. These effects were compared to those of the cyclic AMP analogue 8-(4-chlorophenyl)-thio-cyclic AMP (8-CPTcAMP), which is less susceptible to dephosphorylation. Whereas 8-CPTcAMP almost completely inhibited RNA and DNA synthesis, both 8-chloro-adenosine and 8-chloro-cyclic AMP only partly inhibited synthesis of RNA and DNA at growth-inhibitory concentrations, as demonstrated by using [5-1H] uridine and [14C]thymidine incorporation. Therefore, the growth-inhibitory effect of 8-chloro-cyclic AMP is not (or not completely) due to activation of cyclic AMP-dependent protein kinase nor to the inhibition of nucleic acid synthesis. Flow cytometric analysis revealed that 8-chloro-cyclic AMP and 8-chloro-adenosine probably block cell cycle progression at the G2M phase. The effects of 8-chloro-cyclic AMP on nucleic acid synthesis and cell cycle progression were largely prevented by adenosine deaminase, which inactivates 8-chloro-adenosine. This indicates that the effects of 8-chloro-cyclic AMP were at least in part due to its metabolite 8-chloro-adenosine. Incorporation of 8-chloro-adenosine into RNA and DNA might contribute to the disturbance of the cell cycle kinetics and growth-inhibitory effect of 8-chloro-adenosine.

2-Chloroadenosine↗

PubMed1997

5-fluorouracil/leucovorin-induced inhibition of thymidylate synthase in normal tissues of mouse and man.

We evaluated the effects of 5-fluorouracil (5FU) and leucovorin (LV) on thymidylate synthase (TS) in normal rapidly dividing tissues, which may contribute to toxic side-effects of treatment with 5FU and LV. TS levels were determined in biopsies of human liver and colon mucosa and murine bone marrow, liver and intestinal mucosa at several time points after administration of therapeutic doses of 5FU or LV/5FU. In murine liver, after treatment with 100 mg/kg 5FU, TS inhibition was significantly higher than after LV/5FU administration (P < 0.001). A similar trend was observed in human liver tissue. Murine intestinal mucosa had TS levels below the limit of detection after 5FU or LV/5FU treatment. In human colon mucosa samples, administration of 500 mg/m2 5FU resulted in a large extent of TS inhibition but the small number of samples did not allow a time- or 5FU-LV/5FU-related evaluation. TS activity in murine bone marrow cells was strongly inhibited to 10% of the control value during 48 h. LV/5FU administration resulted in a slightly higher inhibition. No human bone marrow was available to measure TS levels. Both in mice and humans the most pronounced TS inhibition occurred in the tissue that was involved in dose-limiting toxicity. Therefore it is very likely that TS inhibition in normal tissues contributes to the toxic side-effects of 5FU treatment.

Adult↗

PubMed1997

Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and-resistant leukemia cells.

Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.

Adenosine↗

PubMed1997

Biochemical mechanisms of interferon modulation of 5-fluorouracil activity in colon cancer cells.

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.

Animals↗

PubMed1997

Combination chemotherapy studies with gemcitabine.

Gemcitabine (2',2'-difluorodeoxycytidine) is an antineoplastic agent with clinical activity against ovarian carcinoma, small cell and non-small cell lung cancers, head and neck cancer, bladder cancer, breast cancer, and pancreatic cancer. Cisplatin (CDDP), etoposide (VP-16), and mitomycin C (MMC) are well-known anticancer agents that are also active against many of these types of cancer. Because of the low toxicity profile of gemcitabine and the differences in mechanism of cytotoxicity, combinations of these drugs with gemcitabine were studied in vitro and in vivo. Cells were exposed in vitro for 1, 4, 24, or 72 hours to gemcitabine in combination with these drugs, either simultaneously or sequentially in a constant ratio. Another approach consisted of exposure to a combination of the approximate IC25 of one drug and varying concentrations of the other drug. Synergism for several of these combinations was found in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its gemcitabine-resistant variant AG6000, and in the non-small cell lung cancer cell lines H322 and Lewis lung (LL) after a 72-hour drug treatment. Studies of the possible mechanisms of action initially focused on the major metabolic features of each drug. CDDP did not enhance the accumulation of gemcitabine triphosphate and caused only marginal changes in the extent of DNA double-strand breaks (DSBs) induced by gemcitabine in these cell lines. Gemcitabine increased platinum accumulation only in the ADDP cell line, but the DNA platination was enhanced in the A2780, ADDP, AG6000, and LL cell lines. MMC did not influence the formation of DSBs by gemcitabine in the LL cell line. The combination of VP-16 and gemcitabine, however, resulted in the formation of more DSBs in this cell line than each drug alone. This effect was even more pronounced when cells were exposed to VP-16 4 hours before gemcitabine. In vivo, the antitumor activity of a combination of 50 mg/kg gemcitabine and 6 mg/kg CDDP was more effective against LL tumors than each compound alone. In conclusion, gemcitabine is an attractive drug to combine with a wide range of anticancer drugs; synergism is often schedule dependent.

Antimetabolites, Antineoplastic↗

PubMed1997

Modulation of 5-fluorouracil in mice using uridine diphosphoglucose.

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.

Animals↗

PubMed1996

Antitumor activity of prolonged as compared with bolus administration of 2',2'-difluorodeoxycytidine in vivo against murine colon tumors.

2',2'-Difluorodeoxycytidine (gemcitabine) is a cytidine analogue with established antitumor activity against several experimental tumor types and against human ovarian and non-small-cell lung cancer. Both preclinical studies and most clinical trials involving patients with solid tumors have focused on short-term administration schedules; however, mechanistic studies indicate that a continuous-infusion schedule may be more effective. We determined the maximal tolerated dose (MTD) of gemcitabine in mice using various schedules. At these MTDs we observed considerably better antitumor activity of gemcitabine in two of three murine colon carcinoma lines using a prolonged administration as compared with a standard bolus protocol (i.p. 120 mg/kg q3d x 4). On the latter schedule, Colon 26-10 grown in BALB/c mice was the most sensitive tumor line, showing a growth-delay factor (GDF, number of doubling times gained by the treatment) of 6.7, whereas Colon 38 (grown in C57/B16 mice) was the least sensitive tumor, displaying a GDF of 0.9. Prolonged treatment (q3d x 6) of Colon 26-10 at a lower dose (100 mg/kg) enhanced the antitumor activity (GDF 9.6) while producing similar toxicity. A similar weight loss was found following the continuous infusion (c.i.) of gemcitabine using Alzet osmotic pumps s.c. for 3 or 7 days (2 mg/kg), but the GDF increased to 2.4 in Colon 38 (C57/B16) as compared with that provided by the bolus injections. Continuous infusion of gemcitabine at 15 mg/kg per 24 h q7d x 2 i.v. via the tail vein was more effective than bolus injection against Colon 26-10, with the GDF being > 17.7 and 73% of the tumors regressing completely. However, against Colon 38 tumors this schedule was not effective (GDF 0.4), even with a 25% higher dose. The plasma pharmacokinetics of gemcitabine was determined after one bolus dose (120 mg/kg). The peak concentration of gemcitabine was 225 microM and that of the deaminated catabolite 2',2'-difluorodeoxyuridine (dFdU) was 79 microM. The elimination of gemcitabine was much faster than that of dFdU, with the t1/2 beta values being 15 min and 8 h, respectively. For the c.i. schedules, plasma concentrations were below the detection limit of the assay (< 0.5 microM). Our results suggest that prolonged infusion of gemcitabine can give a better antitumor activity than bolus injections and shows promise of being active in clinical trials.

Animals↗

PubMed1996

Tumor size and origin determine the antitumor activity of cisplatin or 5-fluorouracil and its modulation by leucovorin in murine colon carcinomas.

5-Fluorouracil (FUra) is one of the few effective agents in the treatment of patients with colorectal cancer. Its effects on the target enzyme thymidylate synthase (TS) can be modulated by leucovorin (LV) or cisplatin (CDDP). Tumor size and differentiation of tumor characteristics can influence therapeutic efficacy. We therefore studied the relationship between tumor size (cutoff point 200 mm3) and the antitumor activity of FUra and its modulation by LV in murine Colon 26 and Colon 38 tumors. The doubling time of tumors measuring > 200 mm3 was about 160% longer. The antitumor effect of FUra in these large tumors was decreased and could not be modulated by LV. In addition, three subtypes of Colon 26 (Colon 26-A, Colon 26-B, and Colon 26-10) were identified and characterized for tumor-induced weight loss, TS activity, response to chemotherapy, and histological features. Mice bearing Colon 38 and Colon 26-10 did not lose weight as a result of tumor growth. Colon 26-A caused a weight loss of up to 19%, whereas mice with Colon 26-B tumors remained within 10% of their initial weight and tolerated at least 2.5 times more tumor load than did mice bearing Colon 26-A, which induces cachexia. Among untreated tumors, TS catalytic activity was highest in Colon 26-B (5536 pmol mg protein(-1) h(-1)) and lowest in Colon 38 (799 pmol mg protein(-1) h(-1)); Colon 26-A and Colon 26-10 had intermediate activities (about 2500 pmol mg protein(-1) h(-1)). 5-Fluoro-2'-deoxyuridine monophosphate (FdUMP) binding was comparable in the three Colon 26 subtypes but was lower in Colon 38. The antitumor activity of FUra could be modulated by LV in Colon 38, Colon 26-10, and Colon 26-A but could not in Colon 26-B, with complete responses (CR) being obtained in Colon 26-10 and Colon 38. The latter two were highly sensitive to CDDP, followed by Colon 26-A and Colon 26-B (CRs: 50%, 40%, 25%, and 0, respectively). Furthermore, necrosis was noted in Colon 26-B and Colon 38 but not in Colon 26-A. In conclusion, (1) the antitumor activity of FUra in large tumors is decreased and cannot be modulated by LV and (2) characteristics and sensitivity to chemotherapeutics can vary substantially in closely related tumors of the same origin.

Adenocarcinoma↗

PubMed1996

Modulation of metabolism and cytotoxicity of cytosine arabinoside with N-(phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines.

Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells.

Antimetabolites, Antineoplastic↗

PubMed1996

Development, pharmacology, role of DT-diaphorase and prospects of the indoloquinone EO9.

1. The indoloquinone EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2- (1H-indole-4,7-dione)-propenol; E85/053; NSC 382,459) is a synthetic bioreductive alkylating agent that is structurally related to mitomycin C (MMC). 2. EO9 does, however, show a different mechanism of action and a broader antitumour profile than MMC. It is also a more potent cytotoxic agent in vitro than MMC, probably because of its impressive efficient activation by reductive enzymes, particularly DT-Diaphorase. This enzyme is elevated in several tumours compared to normal tissues. 3. The preferential cytotoxicity of EO9 under hypoxic conditions makes it an interesting compound to combine with radiation. 4. In preclinical and the Phase I clinical studies, no myelosuppression was observed but reversible proteinuria was dose-limiting. Phase II clinical studies were started in the summer of 1994.

Animals↗

PubMed1996

18F-radiopharmacokinetics of [18F]-5-fluorouracil in a mouse bearing two colon tumors with a different 5-fluorouracil sensitivity: a study for a correlation with oncological results.

The tissue distribution and biodynamics of 18F-labelled 5-fluorouracil (FU) are described and studied for correlation with its in vivo antitumor activity. The in vivo model consisted of Balb/c mice bearing a FU sensitive (Colon 26-10 carcinoma) tumor in the left and a less responsive (Colon 26 carcinoma) tumor in the right abdominal side of the animal. Distribution and efflux of 18F-label from tumor, blood, and other tissues were determined by obduction at 0.5, 1, 2, 4, and 6 h postintravenous injection. For a comparison, the 18F-labeled 5-fluoro-6-hydroxy and cis-5-fluoro-6-ethoxy uracil adducts were studied in the same in vivo model. For 18F-FU it was found that the 18F-label tumor kinetics rapidly fell into a biphasic mode: a relatively short 18F beta phase (18F t1/2 beta 21 +/- 3 min), linked with the total body metabolic capacity and clearance of the animal, and a longer 18F gamma phase, linked with the intrinsic intratumoral FU metabolism (Colon 26-10: 18F t1/2 gamma 10.3 h; Colon 26: 18F t1/2 gamma 5.6 h). It is proposed that the observed faster 18F efflux of the less responsive Colon 26 corresponds to an enhanced breakdown of 5-fluoronucleotides to 5-fluoronucleosides and subsequent elimination from the tumor cells. It is concluded that on PET scanning, measurement of the dynamic 18F t1/2 gamma and 18F t1/2 beta parameter is of prime importance for an insight in the in vivo tumor biology of a patient.

Animals↗

PubMed1996

Preclinical combination therapy with gemcitabine and mechanisms of resistance.

Cisplatin and gemcitabine both have activity in solid tumors, such as non-small cell lung, ovarian, and head and neck cancers. These drugs have the desired features needed to obtain synergistic activity, different side effect profiles, and mechanisms of action. Cisplatin acts by forming DNA-DNA cross-links (both intrastrand and interstrand) and DNA-protein cross-links; resistance to cisplatin is thought to be due to excision repair of the affected DNA. Gemcitabine acts by its incorporation into nucleic acids, leading to masked chain termination. By combining gemcitabine with cisplatin, it might be possible to achieve a better therapeutic effect than either drug alone and to bypass resistance to one or both drugs. Acquired resistance to gemcitabine was associated with a deoxycytidine kinase deficiency in vitro, but this was difficult to achieve in vivo. Proper scheduling may overcome intrinsic and transient resistance due to physiologic circumstances or aberrant biochemical properties. Preclinical in vitro and in vivo combination studies with cisplatin showed schedule- and model-dependent synergistic and additive effects between cisplatin and gemcitabine. Incorporation of gemcitabine into DNA might facilitate cisplatin-DNA adduct formation. Combining gemcitabine and cisplatin inhibited the DNA excision-repair process more than gemcitabine alone. This implies that if gemcitabine nucleotide is incorporated into the DNA strand, the action of the proofreading exonucleases is less efficient. In addition, both deoxyribonucleotide and ribonucleotide pools, essential for good functioning of DNA repair, are seriously depleted by gemcitabine. It is concluded that combining gemcitabine with cisplatin can be at least additive providing the right schedule is chosen, giving the best balance between acceptable toxicity and an enhanced antitumor activity.

Animals↗

PubMed1996

Synergistic interaction between cisplatin and gemcitabine in vitro.

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.

Antineoplastic Agents↗

PubMed1996

Therapeutic efficacy of fluoropyrimidines depends on the duration of thymidylate synthase inhibition in the murine colon 26-B carcinoma tumor model.

5-Fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) are common chemotherapeutic drugs for the treatment of advanced colorectal cancer. Two recognized mechanisms of action of these agents are inhibition of thymidylate synthase (TS) and incorporation of fluorinated UTP into cellular RNA. In previous studies on drug scheduling of both fluoropyrimidines, we observed the highest therapeutic efficacy by using a weekly i.v. push schedule. Furthermore, weekly 400-mg/kg FdUrd is superior to equitoxic weekly 80-mg/kg FUra in murine Colon 26-B carcinoma. We evaluated the most important pharmacokinetic and pharmacodynamic effects of both fluoropyrimidines to delineate the biochemical mechanisms underlying their differences in therapeutic activity in this tumor model. FUra concentrations and elimination in tumors after FdUrd or FUra administration were comparable, and the level of FUra incorporation into cellular RNA following treatment with FUra or FdUrd was similar. Free tumoral 5-fluoro-dUMP levels were initially 3-fold higher after FdUrd but diminished rapidly thereafter. The number of free [3H]5-fluoro-dUMP-binding sites decreased to about 25 and 15% of control values within 2 h after treatment with equitoxic doses of FUra and FdUrd and remained low for 72 h. The duration of TS inhibition was significantly longer following treatment with FdUrd compared with FUra, 168 and 72 h, respectively. The superiority of the antitumor activity of an i.v. push of FdUrd over FUra in the treatment of Colon 26-B tumors correlates with maintenance of TS inhibition and repeated drug administration when TS remains low, whereas FUra incorporation into RNA does not appear to distinguish the antitumor response of FdUrd from that of FUra in this tumor model.

Animals↗

PubMed1995

Quantification of 5-fluorouracil incorporation into RNA of human and murine tumors as measured with a sensitive gas chromatography-mass spectrometry assay.

5-Fluorouracil (5FU) can exert its cytotoxic activity by either inhibition of thymidylate synthase or incorporation into RNA. The extent and importance of the latter in tumors of patients are not clear, due to the lack of sensitive and reproducible methods. RNA from 5FU-treated human WiDr colon tumor cells was isolated and [(14)CL]5FU incorporation into RNA was measured by traditional scintillation counting while that of nonradiolabeled 5FU was measured with the present, new method. For the latter purpose, isolated RNA was incubated with RNase, alkaline phosphatase, and uridine phosphorylase, resulting in a complete degradation of RNA, nucleotides, and nucleoside to 5FU. 5FU was then measured with gas chromatography coupled to mass spectrometry. For both methods RNA incorporation was 0.4 pmol/h/micrograms RNA at 25 microM 5FU while a similar time (up to 4 h) and concentration dependence (25 to 50 microM) were found. Reproducibility of the assay was more than 95%. In a murine colon tumor 5FU incorporation into RNA reached a peak of 10 pmol/micron RNA at 2 h after administration of the the maximum tolerated dose of 80 mg5FU/kg, which was retained until at least 72 h at 2.5 pmol/micron. In tumors from patients treated with 500 mg5FU/m(2) incorporation into RNA after 24 h amounted to 1.0-1.5 pmol/micrograms RNA. In conclusion, a novel approach, combining different sensitive and reproducible techniques, was established to measure 5FU incorporation into RNA in clinical tumor specimens enabling determination of its clinical relevance.

Animals↗

PubMed1995

Functional activity of the reduced folate carrier in KB, MA104, and IGROV-I cells expressing folate-binding protein.

The role of a membrane-associated folate binding protein (mFBP) in transport of folate analogues was investigated in three epithelial cell lines that were grown in high folate medium and folate-conditioned medium and express different levels of mFBP: human nasopharyngeal KB cells, monkey kidney MA104 cells, and IGROV-I ovarian carcinoma cells. Folate analogues were selected for which mFBP exhibits a low affinity, i.e., methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folic acid, i.e., N-(5[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl(-N-m ethylamino]-2-theonyl)-L-glutamic acid (ZD1694), N10-propargyl-5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies with IGROV-I and MA104 cells demonstrated a lack of correlation between the affinity of mFBP for the antifolate drugs and their sensitivity profile; both cell lines were highly sensitive to growth inhibition by MTX, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were insensitive for CB3717. The same drug sensitivity profile was observed for KB cells, with the exception that these cells were also sensitive to growth inhibition by CB3717 but only in folate-conditioned medium. This overall drug sensitivity profile appeared to correlate with the differential efficiency of drug transport via the "classical" reduced folate/MTX carrier (RFC), rather than by mFBP. Characteristics that further supported functional RFC activity in KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prevented by the reduced folate leucovorin rather than by folic acid; (b) rates for uptake of [3H]10-EdAM were 2-4-fold higher than for [3H]MTX at 1 microM extracellular concentrations and coincided with the affinity of the RFC for these drugs, rather than those of the mFBP; (c) uptake of [3H]10-EdAM and [3H]leucovorin was markedly inhibited by leucovorin and 10-EdAM, respectively, or by an N-hydroxysuccinimide ester of MTX (irreversibly labeling RFC) but only to a minor extent by folic acid or an N-hydroxysuccinimide ester of folic acid (irreversibly labeling mFBP); and, finally, (d) labeling with an N-hydroxysuccinimide ester of [3H]MTX identified a protein with a molecular weight within the range of that reported for the RFC in human leukemic cells. Altogether, these results indicate that both RFC and mFBP are coexpressed in three epithelial cell lines and that RFC is the preferential route of entry for antifolate compounds, even when mFBP is expressed to very high levels.

Aminopterin↗

PubMed1995

Addition of leucovorin in modulation of 5-fluorouracil with methotrexate: potentiating or reversing effect?

Potentiation of the anti-tumor activity of 5-fluorouracil (5FU) by methotrexate (MTX) and leucovorin (LV) requires careful scheduling to achieve the most favorable interactions between these 3 drugs. In vitro the triple combination of MTX/5FU/LV was tested at 4 time intervals and with 3 MTX concentrations in 3 cell lines. MTX/5FU/LV was never superior to both 5FU/LV and MTX/5FU, moreover LV reduced growth inhibition by MTX/5FU in 2 cell lines. MTX/5FU was associated with only moderately increased levels of the 5FU metabolite FdUMP, while LV/5FU reduced the free FdUMP levels. MTX/LV/5FU had a dual effect and FdUMP levels gave no clear indication regarding the anti-proliferative effect that could be expected. In vivo the time-dependent potentiation of MTX on the 5FU anti-tumor activity was confirmed for the treatment of HNX-14C-bearing mice. Addition of LV to the MTX/5FU combination failed to cause a gain in anti-tumor efficacy. For the treatment of Colon-26-bearing mice, MTX/5FU was comparable to LV/5FU, but the triple combination of MTX/LV/5FU seemed to have a better anti-tumor effect. However, the toxicity of the latter treatment was greater than that of LV/5FU. The data from both in vitro and in vivo experiments failed to support the addition of LV to MTX/5FU therapy, possibly due to a reversing effect of LV.

Animals↗

PubMed1995

The combination 5-fluorouracil/levamisole induces enhanced rat Kupffer cell-mediated cytotoxicity in vitro against the syngeneic colon adenocarcinoma cell line CC531.

The mode of action of the combination treatment 5-fluorouracil (5-FU) and levamisole in colorectal cancer patients is unknown. It is postulated that the beneficial effect may be explained by an immunomodulatory effect on Kupffer cell (KC) cytotoxicity. We evaluated the effect of levamisole (200 micrograms/ml) and 5-FU (10 microM) on rat KC cytotoxicity against syngeneic CC531 tumor cells. Viability of KCs was unaffected by 5-FU and/or levamisole. The combination did not enhance growth inhibition of CC531 compared to 5-FU alone. A significant increase in KC cytotoxicity was observed after 24-hr incubation with 5-FU/levamisole especially at an effector/target ratio of 10 (P < 0.05). 5-FU alone had no effect on KC cytotoxicity, while levamisole induced only a slight increase. Our in vitro data suggest that the additive effect of the combination 5-FU/levamisole on KC cytotoxicity may attribute to the beneficial effect of the adjuvant treatment in colorectal cancer patients.

Adenocarcinoma↗