MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5–10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G 1 /S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events.

Myeloid Metaplasia with Myelofibrosis (MMM) is a myeloproliferative disorder (MPD) associating ineffective and extramedullary hematopoiesis with progressive splenomegaly, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of dystrophic megakaryocytes (Mk). We compared the transcriptome of CD34+ and Mk cell from the peripheral blood (PB) of MMM patients and from the PB and bone-marrow (BM) of unmobilized healthy donors. Application of multivariate analyses (principal component analysis and classification by partitioning around medoids algorithm) on Gene Ontology annotation of differential genes allowed a global functionally approach of the main cellular dysregulated pathways in MMM. Each sample cRNA probe was individually and differentially hybridized to the cRNA reference probe on a Human Oligo-microarray 22K (Agilent) and data were normalized by application of the local LOWESS algorithm. Comparison of the CD34+ cell transcriptome between MMM and healthy donors revealed a global down regulation of 2/3 of the expressed genes in contrast to 1/3 of genes that are up-regulated after data filtration by significance analysis microarray (SAM) algorithm (threshold p<0.01). The down-regulation mainly concerns genes involved in hematopoiesis inhibition such as interferon pathway and of lymphoid and apoptotic functions such as BCL6. Such a downregulation is in favour of a lost of survival/proliferation suppression signals rather than of a positive activation process. Up regulated genes included genes linked to Mk differentiation/function, hemostasis and migration process such as tetraspanines and chemokines. In contrast to CD34+ cell, comparison of the transcriptomes from MMM and control Mk revealed an approximate equal number of down- and up-regulated genes after data filtration by Student test algorithm at p-value threshold p<0.05. The gene expression profile of MMM Mk cell exhibited an intense metabolic disturbance (16S proteasome), infidelity of DNA repair compensated by an increase in the control of DNA damage (RINT-1) and possible disturbance of the angiogenic message as show by the down regulation of the TNNI3 troponin. Modulation of these genes might participate in the dysmegakaryopoiesis and the angiogenesis that features MMM. Comparison of CD34+ and Mk cell transcriptomes from patients and healthy donors shows that the two populations exhibit distinct expression profiles. No common differential genes that could be a unique signature of MMM have been identified, confirming the multifactorial and multistep feature of this heterogenous disease. Our data are in agreement with the main pathological features of MMM, especially concerning key genes involved in cell cycle status, proliferation, mobilization and transduction pathways. Furthermore, among these differentially expressed genes, the CD9 tetraspanine and FLT3 genes could be individualized and might represent molecular markers for the diagnosis of JAK2 V617F negative MMM and to distinguish MMM from other MPD.

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

Summary. Deficiency in methylenetetrahydrofolate reductase (MTHFR), the enzyme involved in the remethylation of homocysteine to methionine using methyltetrahydrofolate as cofactor, induces hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and low methylfolate levels. Diagnosis usually occurs during infancy because of various neurological abnormalities. We report MTHFR deficiency diagnosed in an adult woman after a pulmonary embolism. Her adult sister, intellectually retarded, suffered from the same disease. Molecular analysis of the MTHFR gene exhibited four different mutations (two missense mutations, one exon skipping and C677T). The impact of these mutations was analysed through the biological abnormalities in the parents and children.

Methionine depletion in the human cell line CCRF-CEM through the action of recombinant methioninase (rMETase), a methionine-cleaving enzyme, was previously demonstrated to produce a strong cytotoxic synergistic effect with fluorouracil (FUra) throughout a broad range of concentrations of FUra and rMETase, including subcytotoxic levels of rMETase. Potentiation was associated with a decrease in free thymidylate synthase from preexisting levels. To further investigate the action of rMETase on CCRF-CEM cells, in the present study we explored the effects of rMETase as a single agent on DNA methylation levels and DNA synthesis, which may be changed as a result of deprivation of methionine. Cells treated with rMETase under subcytotoxic conditions contained significantly lower levels of genomic methylated DNA than did control cells, as demonstrated by incorporation of the methyl radical of [methyl-(3)H]S-adenosylmethionine in DNA and by use of methylation-sensitive arbitrarily primed PCR. DNA hypomethylation produced by rMETase was of similar magnitude as that produced with the DNA methyltransferase inhibitor 5-azacytidine. Cells exposed to rMETase synthesized significantly more DNA than did untreated cells. Incorporation of [6-3H]thymidine and [6-3H]2'-deoxyuridine in these cells was augmented over that in control by mean factors of 1.78 and 2.36, respectively. Increased 3H nucleoside incorporation resulted in greater numbers of nuclear grains as demonstrated by autoradiography. The increase in DNA synthesis induced by rMETase is likely to result from enhancement of DNA repair because it was not accompanied by differences in cell cycle phase distribution or in total DNA content as determined by flow cytometry. We hypothesize that potentiation of FUra cytotoxicity by rMETase may result from increased inhibition of thymidylate synthase, together with DNA hypomethylation and enhanced DNA repair that could be involved in cell responses to drug-induced damage.

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H4folate) is due to elevation of the methylene tetrahydrofolate (CH2-H4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH2-H4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H4folate) and, subsequently, CH2-H4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H4folate, respectively. 5-HCO-H4folate increased the intracellular concentrations of CH2-H4folate and H4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

Methylenetetrahydrofolate reductase (MTHFR) deficiency was identified in two out of four children born from nonconsanguineous parents. One of the affected children exhibited some clinical findings suggesting cystathionine β-synthase deficiency; MTHFR activity was extremely reduced. In addition, hyperhomocysteinaemia, hypomethioninaemia, low total folate, especially methylfolate in red blood cells, and a reduced methylfolate/total folate ratio were found. Two mutations not yet reported, one on exon 1 of the gene changing an arginine to stop codon and one other on exon 9 changing an arginine to tryptophan were identified in both children in the compound heterozygous state associated with a common polymorphism, 1298A'C, also in the heterozygous state. The mother, homozygous for the mutation on exon 9 and for the polymorphism 1298A'C on exon 7, was clinically and biochemically normal, with normal folate status, mainly methylfolate levels in red blood cells, although MTHFR activity was moderately decreased. The father, heterozygous for the transition arginine to stop codon and for the common polymorphism 677C'T on exon 4, exhibited major biochemical abnormalities, hyperhomocysteinaemia and low methylfolate levels in red blood cells, but was clinically normal. The unaffected children had a biochemical pattern close to that of their mother and were heterozygous for the mutation on exon 9 and also for the two common polymorphisms, 677C'T and 1298A'C.

A diagnosis of methylenetetrahydrofolate reductase (MTHFR) deficiency was made in four sibs at different ages. The first three, including a pair of twins, had retarded psychomotor development, poor social contact, and seizures. Biologically, hyperhomocysteinemia and hypomethioninemia were found associated with low folate levels in serum and red cells, especially undetectable methyltetrahydrofolate in red cells. In the fourth child, prenatal diagnosis was not conclusive because of moderate decrease of enzymatic activity in chorionic villi and trophoblast. The girl was also affected, as shown by hyperhomocysteinemia and low folate levels found several days after birth. A 677C-->T (Ala-->Val) mutation was found in a homozygous state in the four children and in the father. Additionally, a second homozygous mutation, 1081C-->T, changing an arginine to cysteine also was identified in all of the children, whereas the distantly consanguineous parents were heterozygous. This amino acid substitution affecting an arginine residue in a sequence located at the end of catalytic domain seems critical for the function of the enzyme. The difficulty of prenatal diagnosis is discussed given the variability found in enzymatic activity and in the clinical phenotypes.

Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B-12, vitamin B-6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 --> T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 --> T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/-) and normal (-/-) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/-, and 0.71 in -/-. This mutation seems to have an impact on methylTHF generation, These data illustrate the interactions between nutritional and genetic factors.

Prenatal diagnosis for combined methylmalonic aciduria and homocystinuria was performed in five at‐risk pregnancies by determination of methylmalonic acid (MMA) and total homocysteine (Hcy) in amniotic fluid supernatant. The incorporation rate of [14C] propionate (±OHCbl) and the synthesis of cobalamin derivatives in cultured amniocytes were investigated as well as the [14C] MTHF incorporation rate in intact chorion biopsy. Our experience showed that total Hcy and MMA were clearly elevated in amniotic fluid of affected fetuses. Both the study of [14C] propionate incorporation and that of cobalamin synthesis in cultured amniocytes are useful to confirm the results of metabolite determination. The incorporation of [14C] MTHF in intact chorion biopsy seems not to be a reliable diagnostic method. © 1998 John Wiley & Sons, Ltd.

The comparative efficacy of the pure diastereoisomers of leucovorin, the natural (6S) and the unnatural (6R) forms was compared to the racemic form (6RS). A protective effect in methotrexate-treated CCRF-CEM cells was obtained with 6S at concentrations 100-fold higher than those of methotrexate and with 6RS at concentrations 2-fold greater than those of 6S; however, at low concentrations of methotrexate, 6S was more effective than 6RS in preventing the cytotoxicity of methotrexate; on the opposite, 6R exhibited a protective effect at concentrations 10(4) higher than those of methotrexate. On the same cell line, 6S was shown to enhance the cytotoxic effect of 5 Fluorouracil exactly as 6RS while 6R did not exhibit any enhancing effect on cells exposed to 5 Fluorouracil.