Metabolic interaction between methotrexate and 4'-(9-acridinylamino)methanesulfon-M-anisidide in the rabbit

The interaction between methotrexate (MTX) and a new acridine antitumor agent and potent aldehyde oxidase inhibitor, 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA), was investigated both in vivo and in vitro. New Zealand White male rabbits were used for the former experiments under three pharmacokinetic designs: (a) a zero order infusion of mAMSA at 9 mg/h to steady state followed by a single i.v. bolus dose of MTX at 50 mg/kg while maintaining the infusion; (b) a zero order infusion of MTX at 7 mg/h to steady state followed by a single i.v. bolus dose of mAMSA at 5 mg/kg while maintaining the infusion, and (c) a zero order infusion of MTX at 3 mg/h to steady state followed by a zero order infusion of mAMSA at 3 mg/h while maintaining the MTX infusion. In (a) while the mean AUC for MTX (15815 +/- 1317 microMmin) with mAMSA (+mAMSA) remained essentially unchanged relative to that without mAMSA (-mAMSA) at the same dose (14832 +/- 5151 microMmin), the mean AUC of the metabolite 7-hydroxymethotrexate (7-OH MTX) decreased from 9338 +/- 3057 (n = 6, -mAMSA) to 5794 +/- 1371 microMmin (n = 6, +mAMSA). Urinary excretion of 7-OH MTX also decreased from 40.3 +/- 9.5% (n = 6) (-mAMSA) to 23.8 +/- 6.1% dose (n = 6) (P less than 0.01) (+mAMSA) in 8 h with essentially no change in MTX excretion. The fractional rate conversion of MTX to this metabolite (fmi) also decreased from 0.60 +/- 0.19 (n = 6) to 0.40 +/- 0.10 (n = 6) (P less than 0.05). No change in terminal half-lives of MTX and 7-OH MTX was apparent. In (b) MTX steady state levels increased with the concomitant decrease in 7-OH MTX levels in the presence of mAMSA such that their concentration ratios (7-OH MTX/MTX) decreased to 43, 54, 75, and 76% of the pre-mAMSA values, respectively, in four rabbits. In the presence of mAMSA, clearance of MTX at steady state decreased significantly relative to those without mAMSA. Similar results were also observed in (c) except that the perturbation of MTX metabolism was more profound consistent with the experimental setting. No change in protein binding of MTX or the metabolite was apparent in the presence of mAMSA. Rabbit liver homogenate was used in the in vitro experiments which yielded a classical competitive inhibition on the double-reciprocal plot when conversion of MTX to 7-OH MTX was monitored.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine and methotrexate

 Clinical and experimental pharmacokinetic interaction between 6-mercaptopurine (6-MP) and methotrexate (MTX) was investigated in patients as well as in rats and in HL-60 human leukemic cells. Ten children affected by acute lymphoblastic leukemia (ALL) in remission received daily doses of 6-MP given at 25 mg/m² and i.v. infusion of high-dose MTX at 2 or 5 g/m² once every other week. When 6-MP was given alone, the mean peak plasma concentration (C_max) and area under the curve (AUC) of 6-MP were 72.5 ng/ml and 225.3 h ng ml^-1. Concurrent treatment with MTX at 2 or 5 g/m² resulted in a mean increase of 108% and 121% in the C_max and of 69% and 93% in the AUC, respectively. In rats treated with an oral dose of 6-MP at 75 mg/m², MTX given i.p. at 5 g/m² produced mean increases of 110% and 230% in the C_max and AUC of 6-MP, respectively. In HL-60 human leukemic cells incubated with 6-MP at 250 ng/ml, the cumulative intracellular concentration of 6-thioguanine and 6-MP nucleotides was not significantly modified by treatment with 20 μg/ml of MTX. The present findings indicate that high-dose MTX enhances the bioavailability of 6-MP as evidenced by the observed increases in the plasma C_max and AUC of 6-MP in humans and animals. Received: 13 December 1994/Accepted: 12 July 1995     

Biochemical and pharmacologic rationale for high-dose methotrexate

High-dose methotrexate (HDMTX) regimens were initially designed to overcome methotrexate (MTX) resistance due to defective transport of the drug. At high concentrations, enough MTX diffuses into resistant cells to saturate and inhibit the target enzyme, dihydrofolate reductase (DHFR). The high doses of MTX needed to achieve these high drug concentrations must be administered with the reduced folate antidote, leucovorin (LV; 5-formyltetrahydrofolate), to prevent increased toxicity. To increase MTX therapeutic index, LV rescue must be selective, i.e., more effective in normal than in tumor cells. In experimental models, selective rescue can be achieved if strict LV administration guidelines are respected. Since both MTX and LV use the membrane transport system, it was hypothesized that selective rescue occurred because transport-deficient, MTX-resistant tumor cells also transported LV poorly. The unsatisfactory clinical results frequently obtained with HDMTX regimens suggest a need to re-evaluate this underlying rationale, especially in view of recent findings concerning the mechanisms of MTX resistance and LV rescue. Experimentally, cells resistant to MTX because of an increased or altered DHFR, decreased metabolism to polyglutamates, or decreased thymidylate synthase activity are not always significantly more sensitive to higher concentrations of MTX. Furthermore, recent studies on human small cell lung cancer cell lines suggest that decreased MTX polyglutamate metabolism and thymidylate synthase activity might be prevalent MTX-resistant mechanisms in human tumors. Selective LV rescue could also occur through mechanisms other than selective uptake by normal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and pharmacological effects of toremifene metabolites

Summary Toremifene, a new antiestrogenic antitumor compound, has several biologically active metabolites. The hormonal effects of the main metabolites resemble those of unchanged toremifene. The main metabolite in humans,N-demethyltoremifene, is bound to estrogen receptors (ER), inhibits the growth of MCF-7 cells, and exerts an antiestrogenic effect similar to that of toremifene. However, its antitumor effect in vivo against dimethyl-benz(a)anthracene (DMBA)-induced rat mammary cancers is weaker than that of toremifene. Didemethyltoremifene has antiestrogenic actions in mouse and rat uterus at high doses. 4-Hydroxytoremifene is bound to ER with higher affinity and inhibits MCF-7 growth at concentrations lower than those of toremifene. It has a weaker intrinsic estrogenic effect than does toremifene. The efficacy of 4-hydroxytoremifene against DMBA-induced cancers is weak except at very high doses. Oxidations ofN-demethylated metabolites to (deamino)hydroxylated compounds and carboxylic acids are the detoxification routes of toremifene. (deaminohydroxy)Toremifene has only weak hormonal actions at high doses and carboxylated metabolites have no estrogenic/antiestrogenic effects. The antitumor effect of toremifene in vivo is mainly due to unchanged toremifene, but hormonal effects (which may have a role in antitumor actions) are partly attributable to metabolitesN-demethyltoremifene, didemethyl-toremifene, (deaminohydroxy)toremifene, 4-hydroxy-N-demethyltoremifene, and 4-hydroxytoremifene, which have pharmacological properties similar to those of toremifene.     

Accumulating evidence for a drug-drug interaction between methotrexate and proton pump inhibitors

Background.

A number of medications are known to interact with methotrexate through various mechanisms. The aim of this article is to apprise practitioners of a new labeling change based on the accumulating evidence for a possible drug–drug interaction between methotrexate (primarily at high doses) and proton pump inhibitors (PPIs).

Methods.

The U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) database of spontaneous adverse event reports and the published literature were searched for cases reporting an interaction between methotrexate and PPIs.

Results.

A search of the AERS database and existing literature found several individual case reports of drug–drug interactions and three additional supportive studies that suggest potential underlying mechanisms for the interaction.

Conclusion.

There is evidence to suggest that concomitant use of methotrexate (primarily at high doses) with PPIs such as omeprazole, esomeprazole, and pantoprazole may decrease methotrexate clearance, leading to elevated serum levels of methotrexate and/or its metabolite hydroxymethotrexate, possibly leading to methotrexate toxicities. In several case reports, no methotrexate toxicity was found when a histamine H₂ blocker was substituted for a PPI. Based on the reviewed data, the FDA updated the methotrexate label to include the possible drug–drug interaction between high-dose methotrexate and PPIs. Physicians should be alerted to this potential drug–drug interaction in patients receiving concomitant high-dose methotrexate and PPIs.     

Effect of interaction between methotrexate and dihydrofolate reductase on DNA synthesis in L1210 cells in vitro

L1210 leukaemia cells were preloaded with [³H methotrexate] (MTX) to saturate high-affinity intracellular sites, and were then incubated with [³H]MTX to determine the steady-state intracellular MTX concentrations at extracellular concentrations ranging from 10 μM to zero. In addition, incubations to generate incomplete saturation of high-affinity intracellular MTX-binding sites were also carried out. Following determination of the total intracellular MTX, cells were pulsed with deoxyuridine (UdR) and its incorporation into DNA examined to assess the role of exchangeable and bound intracellular MTX on DNA synthesis. Further, cell pellets were disrupted and dihydrofolate reductase (DHFR) activity determined under each experimental condition. Extracellular MTX concentrations in excess of 1 μM depressed UdR incorporation to <2% of control, but incorporation rapidly recovered to 62% of control at the point of MTX-DHFR equivalence, and exceeded control values when all high-affinity sites were not saturated. DHFR activity was undetectable at extracellular MTX concentrations >0.50 μM, and never exceeded 6.09% of control at the “equivalence point” where all high-affinity sites were saturated. When less than 10% of potential inhibitor sites were occupied, enzyme activity increased rapidly, but never reached control. However, 5% of the DHFR activity was sufficient to permit UdR incorporation to continue at 50% of control levels, and UdR incorporation returned to control levels at 20% of the DHFR activity. The relationship between cellular MTX content and DNA synthesis or DHFR activity is sigmoid, suggesting a reversible interaction between enzyme and inhibitor. This lends support to the notion that “free” intracellular MTX is necessary for a maximal antitumour effect, and may explain its role in “high-dose” MTX therapy in man.     

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 M for MTX alone versus 0.088 M for the 1:1 mixture with 7-OH MTX, respectively (p0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.     

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.     

Biochemical and pharmacokinetic effects of leucovorin after high-dose methotrexate in a murine leukemia model.

The administration of calcium leucovorin, either concurrently or after high dosages of methotrexate in L1210 leukemic mice, has both pharmacokinetic and biochemical effects in tumor cells and drug-limiting proliferative normal tissue in small intestine. A reduction in the maximal level of accumulation and retention of exchangeable drug (unbound to dihydrofolate reductase) in tissue could be demonstrated when calcium leucovorin was given simultaneously with methotrexate at equal or greater dosages than the latter. The dose dependence for calcium leucovorin-introduced drug loss is similar in both tissues and showed the expected variation when the time interval between methotrexate and calcium leucovorin doses was increased. With 400 mg methotrexate per kg, greater than 96 mg calcium leucovorin per kg were required maximally to affect overall drug retention in tissue 2 hr after drug, whereas only 24 mg calcium leucovorin per kg were required 16 hr after drug. Calcium leucovorin, given after methotrexate, induced synchronous recovery of DNA synthesis (measured by labeled deoxyruridine incorporation) in both small intestine and L1210 cells. An initial cycle of synthesis was induced in the presence of exchangeable levels of drug. Two hr after methotrexate, 12 mg/kg, calcium leucovorin induced an immediate but only partial (20 to 25% of control rate) recovery of synthesis with dose dependence from 3 to 12 mg calcium leucovorin per kg. Less synthesis was induced after 96 mg/kg and almost none after methotrexate, 400 mg/kg. With calcium leucovorin, 24 mg/kg, given 2 hr after methotrexate, 12 or 96 mg/kg, a major cycle of synthesis occurred when total drug levels approached the equivalence of the dihydrofolate reductase content. The magnitude of this cycle of synthesis in both L1210 cells and small intestine was the same as that seen in animals recovering from methotrexate alone. However, this is based on the assumption that an approximately equivalent relationship between DNA synthesis and labeled doexyuridine incorporation in each tissue during the period of maximal incorporation within the cycle. The major effect of calcium leucovorin, then, was to induce an earlier resumption of DNA synthesis as a consequence of the pharmacokinetic effect in each tissue. With calcium leucovorin, 24 or 400 mg/kg, given 16 hr after methotrexate, an identical effect on drug retention was observed in both L1210 cells and small intestine. Although there was a difference in the time course for recovery in small intestine at each dosage of calcium leucovorin, the recovery of DNA synthesis as drug levels approached the dihydrofolate reductase content was similar in magnitude. In L1210 cells, however, substantial recovery of synthesis to a comparable level and with a similar time-course occurred only after leucovorin, 400 mg/kg. Little or no recovery of DNA synthesis was observed after calcium leucovorin, 24 mg/kg, during the same time period. This dosage schedule (methotrexate, 400 mg/kg, s.c...     

The interaction between fluoropyrimidines and methotrexate,and [14C]-formate incorporation into nucleic acids and protein

Summary Changes are reported in [¹⁴C]-formate incorporation into nucleic acids and protein of Ehrlich ascites tumor cells during exposure to methotrexate (MTX) and fluoropyrimidines. The rate of [¹⁴C]-formate incorporation into RNA, DNA, and protein in the presence of only MTX was inhibited by 82%, 91%, and 75% respectively, when compared with control rates. However, in the presence of 5-fluorodeoxyuridine (FdUrd) plus MTX, formate incorporation into RNA, DNA, and protein was inhibited by 67%, 85%, and 66%. Incubation of cells in vitro with [³H]-dihydrofolate (DHF) results in its rapid conversion to [³H]-tetrahydrofolate (THF). The THF/DHF ratio from the soluble fraction of cells that were incubated with [³H]-DHF was 43% greater in the presence of FdUrd and MTX than in the presence of MTX alone. As the rate of [³H]-dUrd incorporation into DNA was reduced by 88% and 99% by pretreating cells with 0.1 M and 1 M FdUrd, respectively, the inhibitory effect of MTX on [¹⁴C]-formate incorporation into (a) RNA was decreased by 63% and 46%; (b) DNA was decreased by 74% and 61%; and (c) protein was decreased by 63% and 32%. These data suggest that fluoropyrimidines can antagonize the effects of MTX on purines or nucleic acid synthesis and protein synthesis by preventing the consumption of THF for dTMP synthesis.The abbreviations used are MTX methotrexate - FdUrd 5-fluorodeoxyuridine - Fura 5-fluorouracil - FdUMP 5-fluorodeoxyuridine monophosphate - dUrd deoxyuridine - dUMP deoxyuridine monophosphate - dTMP thymidylate - DHF dihydrofolate - THF tetrahydrofolate - 5,10-CH₂THF 5,10-methylene tetrahydrofolate - DHFR dihydrofolate reductase     

Altered pharmacokinetics and clinical consequences of low dose methotrexate plus cisplatin in the treatment of advanced head and neck cancer

Thirty-two patients with head and neck carcinoma received a multidrug chemotherapy protocol including low dose methotrexate (LDMTX) (30 mg/m²) and cisplatin as their initial treatment. A sensitive immunoenzymatic technique was used for systematic MTX blood monitoring (0–56 hr) in all patients. The MTX-related side effects observed in 15 patients (47%) were significantly associated with an increase in systemic drug exposure occurring early during drug infusion. The average end-of-infusion concentration varied from 8 × 10⁻⁷ M for nontoxic patients to 1.45 and 3.12 10⁻⁶ M for moderately and severely toxic patients respectively. The area under the curve (AUC) (0–56 hr) was also directly related to the increase in side-effects. Total body clearance was reduced in an inverse manner. Volumes of distribution and terminal elimination half-lives were not related to the presence or intensity of MTX side-effects. Based on these data, the institution of folinic acid rescue adapted to the MTX blood concentration, a measure previously not suggested for LDMTX, completely prevented severe toxicity in a subsequent series of 26 patients without modification of the response rate.     

Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.     

Biochemical and growth inhibitory effects of the erythro and threo isomers of gamma-fluoromethotrexate, a methotrexate analogue defective in polyglutamylation

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.     

Biochemical consequences of 5-fluorouracil gastrointestinal toxicity in rats; effect of high-dose uridine

Selective protection of the normal host tissues from the toxic effects of anticancer agents would allow the use of higher, probably more effective, doses of the drugs. It has been demonstrated that delayed high-dose uridine administration after 5-fluorouracil decreases the extent of myelosuppression and causes faster regeneration of the bone marrow. We studied the biochemical consequences of the gastrointestinal toxicity caused by 5-fluorouracil and the potential of high-dose uridine treatment to influence these adverse effects. 5-Fluorouracil caused dose-related decreases in the biochemical parameters (thymidine kinase, sucrase, maltase, alkaline phosphatase) selected as early markers of the impaired metabolic activity of the intestinal mucosa. The nadir of the biochemical changes was reached between 24 h and 72 h after 5-fluorouracil treatment, and complete regeneration of the mucosa took 6–7 days. Delayed high-dose uridine administration failed to mitigate the severity of the gastrointestinal damage that ensued after 5-fluorouracil treatment, but caused significantly earlier regeneration of the mucosa.     

Modelling the consequences of interactions between tumour cells.

Classical models of tumorigenesis assume that the mutations which cause tumours to grow act in a cell-autonomous fashion. This is not necessarily true. Sometimes tumour cells may adopt genetic strategies that boost their own replication and which also influence other cells in the tumour, whether directly or as a side-effect. Tumour growth as a whole might be enhanced or retarded. We have used mathematical models to study two non-autonomous strategies that tumour cells may use. First, we have considered the production by tumour cells of an angiogenesis growth factor that benefits both the cell from which it originates and neighbouring cells. Second, we have analysed a situation in which tumour cells produce autocrine-only or paracrine-only growth factors to prevent programmed cell death. In the angiogenesis model, stable genetic polymorphisms are likely to occur between cells producing and not producing the growth factor. In the programmed cell death model, cells with autocrine growth factor production can spread throughout the tumour. Production of paracrine-only growth factor is never selected because it is ''altruistic'' (that is of no benefit to the cell that makes the growth factor), despite being potentially beneficial to tumour growth as a whole. No polymorphisms can occur in the programmed cell death model. Production of angiogenesis and other growth factors in tumours may be under stable genetic, rather than epigenetic, control, with implications for therapies aimed at such targets. Many of the mutations observed in tumours may have non-autonomous effects.