Francis M. Sirotnak

RFC-1 gene expression regulates folate absorption in mouse small intestine

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l,L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and highly saturable (Km or Ki = 2 to 4 μM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 μM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l,L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.

Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics

Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics.

Randomized Phase II Study of Pulse Erlotinib Before or After Carboplatin and Paclitaxel in Current or Former Smokers With Advanced Non–Small-Cell Lung Cancer

PURPOSE A prior study demonstrated that addition of continuous daily erlotinib fails to improve response rate or survival in non-small-cell lung cancer (NSCLC) patients treated with carboplatin and paclitaxel. However, preclinical data support the hypothesis that intermittent administration of erlotinib before or after chemotherapy may improve efficacy. We tested this hypothesis in patients with advanced NSCLC. PATIENTS AND METHODS Eligible patients were former or current smokers with chemotherapy-naive stage IIIB or IV NSCLC. All patients received up to six cycles of carboplatin (area under the curve = 6) and paclitaxel (200 mg/m(2)), with random assignment to one of the following three erlotinib treatments: erlotinib 150 mg on days 1 and 2 with chemotherapy on day 3 (150 PRE); erlotinib 1,500 mg on days 1 and 2 with chemotherapy on day 3 (1,500 PRE); or chemotherapy on day 1 with erlotinib 1,500 mg on days 2 and 3 (1,500 POST). The primary end point was response rate. RESULTS Eighty-six patients received treatment. The response rates for the 150 PRE, 1,500 PRE, and 1,500 POST arms were 18% (five of 28 patients), 34% (10 of 29 patients), and 28% (eight of 29 patients), respectively. The median overall survival times were 10, 15, and 10 months for the 150 PRE, 1,500 PRE, and 1,500 POST arms, respectively. The most common grade 3 and 4 toxicities were neutropenia (39%), fatigue (15%), and anemia (12%). Grade 3 and 4 rash and diarrhea were uncommon. CONCLUSION Patients treated on the 1,500 PRE arm had the highest response rate and longest survival, with ranges similar to those reported for carboplatin, paclitaxel, and bevacizumab in a more restricted population. Further evaluation of this strategy in a phase III trial is proposed.

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.

Posttranscriptionally Mediated Decreases in Folylpolyglutamate Synthetase Gene Expression in Some Folate Analogue-resistant Variants of the L1210 Cell EVIDENCE FOR AN ALTERED COGNATE mRNA IN THE VARIANTS AFFECTING THE RATE OF DE NOVO SYNTHESIS OF THE ENZYME

L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to −7 and L1210/EDX-12 to −14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Vmax with no change in apparent Km. The results of an analysis of FPGS activity in mixtures of variant and parental cell extract suggested that no endogenous inhibitors in the variant cells or stimulatory factors in parental cells accounted for the differences observed. Also, FPGS from variant and parental cells showed no difference in thermostability. Decreases in a 60-61-kDa protein as shown by immunoblotting with anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compared with parental cells. However, no evidence was obtained for a difference in turnover of FPGS protein during measurement of the decay of FPGS activity in cycloheximide-treated variant and parental cells. In addition, Northern blotting of poly(A)+ RNA did not reveal any difference in the size or level of FPGS mRNA among these various cell types. Studies of in vitro translation of hybridization-selected FPGS mRNA from L1210 cells showed that both mitochondrial and cytosolic forms of FPGS were generated during the reaction. Moreover, FPGS mRNA from the variant cells was significantly less effective in mediating formation of the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types. These results suggest that FPGS gene expression in these variants is posttranscriptionally altered at the level of the cognate mRNA itself and that this alteration constitutively down-regulates the steady-state level of FPGS in these variants.

Growth inhibitory, transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Abstract Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.

Phase II Trial of Pralatrexate (10-Propargyl-10-deazaaminopterin, PDX) in Patients with Unresectable Malignant Pleural Mesothelioma

Background: Several previous clinical trials have shown that malignant pleural mesothelioma is responsive to antifolates. The dihydrofolate reductase inhibitor, pralatrexate, has a favorable toxicity profile, primarily limited to stomatitis, and has demonstrated activity in patients with non-small cell lung cancer. In mesothelioma cell lines and xenografts, pralatrexate demonstrated significant antitumor activity. Methods: We conducted this phase II study to determine the response rate of malignant pleural mesothelioma to pralatrexate at a dose of 135 mg/m2 IV every 2 weeks. After a protocol amendment, patients were supplemented with vitamin B12 and folic acid at the time of starting therapy. Results: A total of 16 assessable patients were enrolled. No complete or partial responses were observed. Two patients with epithelioid histology had minor responses. Three other patients remained on study with stable disease for 9, 9, and 48 months. The median time to progression was 3 months. The overall median survival time was 7 months (95% confidence interval: 3.2–16.2 months) and the one-year survival was 31% (95% confidence interval: 15%–65%). Three patients (19%) had grade 2 stomatitis, eight (50%) had grade 3, and one (6%) had grade 4. Conclusions: With this particular dose and schedule, pralatrexate as a single agent had no activity in malignant pleural mesothelioma.

Contrasting effects of oncogene expression on two carrier-mediated systems internalizing folate compounds in Fisher rat 3T3 cells.

Folate compound transport into Fisher rat 3T3 (FR3T3) cells at physiological pH occurs predominantly by an acid pH‐dependent, mobile carrier system. However, influx of [3H]MTX by this system is 3–4‐fold higher at pH 6 than at pH 7.5, the optimum for RFC‐1–mediated folate compound transport. This acid pH dependency reflects an alteration of influx Vmax rather than of influx Km in these cells at different pH. Acid pH‐dependent folate compound transport interacts effectively with MTX, 5ℓLCHO‐folateH4, 5ℓLCH3‐folateH4 and folic acid as permeants (influx Ki = 2.7–5.3 μM). The relative inhibition of influx of [3H]MTX by the organic anions, probenecid, and PO4 was different than for RFC‐1 mediated influx. The folate requirements for growth in culture of FR3T3 cells and cytotoxicity of MTX compared to L1210 cells reflects the interactions of these folate compounds with acid pH‐dependent folate transport. 5ℓLCHO‐folateH4 and PO4 act as exchange anions for this system but their transpositioning has variable effects on transport. 5ℓLCHO‐folateH4 inhibits influx (decelerative equilibrium exchange) but stimulates efflux of [3H]MTX (accelerative equilibrium exchange) while PO4 inhibits efflux. In FR3T3 cells transfected with cmyc and Hras, influx Vmax for [3H]MTX is downregulated 4‐fold and 9‐fold, respectively. At the same time, RFC‐1 expression, which is detectable in FR3T3 cells at the level of its mRNA and RFC‐1 mediated folate compound transport, is increased 3–5‐fold in these transfectants. The increase in RFC‐1 expression in FR3T3Hras cells appears to result from a higher rate of transcription of the gene in these cells as determined by a luciferase reporter gene assay of RFC‐1 promoter activity. This downregulation of the acid pH dependent system and concomitant upregulation of the RFC‐1 mediated system markedly altered pH dependency for influx of [3H]MTX in these transfectants compared to that seen in untransfected cells. We conclude that the major route for internalization at a physiological pH of folate compounds in FR3T3 cells is by an acid pH‐dependent carrier‐mediated system independent of RFC‐1 expression and is downregulated by oncogene expression. J. Cell. Physiol. 184:364–372, 2000.

Different Antifolate-resistant L1210 Cell Variants with either Increased or Decreased Folylpolyglutamate Synthetase Gene Expression at the Level of mRNA Transcription

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H.(1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of l,L5-CHO-folateH (natural diastereoisomer of 5-formyltetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in K and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constituitively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Additional Organizational Features of the Murine Folylpolyglutamate Synthetase Gene TWO REMOTELY SITUATED EXONS ENCODING AN ALTERNATE 5′ END AND PROXIMAL OPEN READING FRAME UNDER THE CONTROL OF A SECOND PROMOTER

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5′ end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5′-untranslated region. Exon A1b encodes a downstream segment of the 5′-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5′ to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3′ end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.