Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line.

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.

Expression of Escherichia coli methionyl-tRNA formyltransferase in Saccharomyces cerevisiae leads to formylation of the cytoplasmic initiator tRNA and possibly to initiation of protein synthesis with formylmethionine.

Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.

Expression analysis of human HL60 cells exposed to 60 Hz square- or sine-wave magnetic fields.

A total of 960 complementary DNA (cDNA) clones from an HL60 cell cDNA library were screened to discover genes that were differentially expressed in HL60 cells exposed to 60 Hz square-wave magnetic fields (MFs) compared to sham-exposed cells. Square-wave fields are rich in odd harmonic frequency content. We used a two-gel cDNA library screening method (BIGEL) to identify treatment-induced alterations in gene expression. Four cDNA clones were tentatively identified as differentially expressed after exposure to square-wave MFs at 2 mT for 24 h. BIGEL-identified genes (GenBank accession number) corresponding to these clones were: TI227H (D50525), EST Homo sapiens partial cDNA (Z17814), human ribosomal protein S13 (L01124), and AICAR transformylase mRNAs (D82348). The differences in mRNA levels were not confirmed in test compared to experimental cells by Northern analysis. In other experiments, we used concurrent exposure to 60 Hz sine- or square-wave MFs (0 or 2 mT, duration of 3 or 24 h, no postexposure delay). In addition to the four BIGEL genes, we also investigated MYC, HSP70, RAN and SOD1. In the case of MYC and HSP70, square-wave MFs appeared to exhibit more marked alterations when compared to sinusoidal waveforms, but the overall results indicated no effect of possible differential magnetic-field-induced expression of all eight genes. In contrast, alterations of mRNA levels were observed for seven genes after exposure to X irradiation, hyperthermia and TPA. These results are contrary to previously proposed similarities between the action of these agents and MF effects on gene transcription.

Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis.

The non-Hodgkin lymphoma (NHL) subtype anaplastic large-cell lymphoma (ALCL) is frequently associated with a t(2;5)(p23;q35) that results in the fusion of the ubiquitously expressed nucleophosmin (NPM) gene at 5q35 to the anaplastic lymphoma kinase (ALK) gene at 2p23, which is not normally expressed in hematopoietic tissues. Approximately 20% of ALCLs that express ALK do not contain the t(2;5), suggesting that other genetic abnormalities can result in aberrant ALK expression. Here we report the molecular characterization of an alternative genetic means of ALK activation, the inv(2)(p23q35). This recurrent abnormality produces a fusion of the amino-terminus of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional homodimeric enzyme that catalyzes the penultimate and final steps of de novo purine nucleotide biosynthesis, with the intracellular portion of the ALK receptor tyrosine kinase. RT-PCR analysis of 5 ALCL tumors that contained the inv(2) revealed identical ATIC-ALK fusion cDNA junctions in all of the cases. Transient expression studies show that the ATIC-ALK fusion transcript directs the synthesis of an approximately 87-kd chimeric protein that is localized to the cytoplasm, in contrast to NPM-ALK, which typically exhibits a cytoplasmic and nuclear subcellular distribution. ATIC-ALK was constitutively tyrosine phosphorylated and could convert the IL-3-dependent murine hematopoietic cell line BaF3 to cytokine-independent growth. Our studies demonstrate an alternative mechanism for ALK involvement in the genesis of NHL and suggest that ATIC-ALK activation results from ATIC-mediated homodimerization. In addition, expected decreases in ATIC enzymatic function in ATIC-ALK-containing lymphomas may render these tumors more sensitive to antifolate drugs such as methotrexate. (Blood. 2000;95:2144-2149)

Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli.

The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res. 22, 2476-2478]. Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized. The enzyme catalyzed specific methylation of C967 of unmodified 16S rRNA transcripts. A 16mer stem-loop structure containing C967 (nt 960-975) was also a good substrate for the enzyme in vitro. Methylation of C967 was confirmed by several methods including analysis of RNase T1 digests and nearest-neighbor analysis. Fmu did not catalyze methylation of transcripts of 23S rRNA. E. coli cells that contained kanr-disrupted fmu produced 16S rRNA that could be specifically methylated by Fmu in vitro at C967 but not C1407. Further, fmu disruption did not significantly alter the growth rate of E. coli in rich or minimal media. We propose renaming this ORF "rrmB" and the enzyme "RrmB" for rRNA methyltransferase.

X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli.

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second step of the de novo purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. GAR-syn containing an N-terminal polyhistidine tag was expressed as the SeMet incorporated protein for crystallographic studies. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multiple-wavelength anomalous diffraction (MAD) phase determination and refined to 1.6 A resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas the smaller B domain is extended outward. Two hinge regions, which might readily facilitate interdomain movement, connect the B domain and the main core. A search of structural databases showed that the structure of GAR-syn is similar to D-alanine:D-alanine ligase, biotin carboxylase, and glutathione synthetase, despite low sequence similarity. These four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-binding enzyme with low sequence similarity but unknown function, synapsin Ia, was also found to share high structural similarity with GAR-syn. Interestingly, the GAR-syn N domain shows similarity to the N-terminal region of glycinamide ribonucleotide transformylase and several dinucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of these models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP binding belong to the A or B domains. The N and C domains appear to be largely responsible for substrate specificity. The structure of GAR-syn allows modeling studies of possible channeling complexes with PPRP amidotransferase.