Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro.

BACKGROUND AND PURPOSE: Both CB(1) and CB(2) cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro. EXPERIMENTAL APPROACH: Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α(v) ß(3) or with F-actin rings, or measurement of resorption area. KEY RESULTS: Human osteoclasts express both CB(1) and CB(2) receptors. CB(2) expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB(1) and/or CB(2) antagonists. CONCLUSIONS: AND IMPLICATIONS Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB(2) receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Regulation of dihydropyrimidine dehydrogenase in colorectal cancer.

Dihydropyrimidine dehydrogenase (DPD) is responsible for degradation of the pyrimidines uracil and thymine and the inactivation of the chemotherapeutic agent 5-fluorouracil. DPD activity is highly variable in cancer populations, and this variation may influence the antitumor efficacy of 5-fluorouracil. However, little is known about the regulation of DPD mRNA expression in any tissues. Using a reverse transcription competitive PCR assay, we quantified DPD mRNA levels in 10 matched colorectal tumors and adjacent normal mucosae and 7 colorectal liver metastases and adjacent normal livers. Lower levels of DPD mRNA expression were observed in colorectal tumor compared with adjacent normal colon mucosa (median, 0.01 versus 0.37 amole/microg total RNA, P = 0.02). DPD mRNA expression was also lower in metastases than adjacent normal liver tissue (median, 0.11 versus 1.17 amole/microg total RNA, P = 0.001). DPD mRNA expression was higher in normal liver than normal colonic mucosa (median, 1.17 versus 0.37 amole/microg total RNA, P = 0.02). A significant relationship was observed between DPD mRNA and catalytic activity (r(s) = 0.66, P<0.001). The tumor:normal ratio for DPD mRNA, protein, and activity was relatively stable in liver (0.25, 0.55, and 0.51, respectively) but varied considerably in colon (0.085, 0.9, and 1.25, respectively), consistent with enhanced translation of DPD transcript in primary colorectal tumor. This suggests that DPD can be regulated at the levels of both transcription and translation.

Dihydropyrimidine dehydrogenase pharmacogenetics in Caucasian subjects.

AIMS: Dihydropyrimidine dehydrogenase (DPD) catalyses the reduction of pyrimidines, including the anticancer agent 5-fluorouracil (5FU). Impaired 5FU degradation, through low DPD activity, has led to severe, life-threatening or fatal toxicity after administration of 5FU. Complete DPD deficiency is associated with the inherited metabolic disease thymine uraciluria. Several mutations in the gene encoding DPD have recently been identified, but the phenotype-genotype concordance of these alterations in the general population has not been reported. METHODS: Mononuclear cells were isolated from whole blood and DPD activity was determined after ex vivo incubation with 14C-5FU followed by h.p.1.c. analysis of 5FU metabolites. Analysis of mutations in the DPD gene at an exon splice site, codons 534, 543, and 732, and a deletion at base 1897 (deltaC1897) were performed in 30 subjects with the lowest and 30 subjects with the highest enzyme activity using PCR-RFLP. RESULTS: DPD activity was measured in 226 Caucasian subjects and was highly variable (range 19.1-401.4 pmol min(-1)mg(-1) protein). Mutations were frequently observed at codons 543 (allele frequency 28%), 732 (allele frequency 5.8%), and 534 (allele frequency 0.8%), but were not associated with low DPD activity. There were no splice site or deltaC1897 mutations found in this population. CONCLUSIONS: The five mutations analysed in this study are insufficient for identification of patients at risk for 5FU toxicity or thymine uraciluria. Both the splice site mutation and deltaC1897 are relatively rare in the general Caucasian population. Therefore, identification of further molecular alterations is required to facilitate the use of DPD analysis in genetic diagnosis and cancer therapeutics.

The second ETV6 allele is not necessarily deleted in acute leukemias with a ETV6/ABL fusion.

The ETV6 (TEL) locus at chromosome band 12p 13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often deleted. In addition, loss of heterozygosity of ETV6 is frequently observed in childhood'ALL. Thus, it has been suggested that ETV6 may have an anti-oncogenic role to play, in addition to its oncogenic role. We have described an unusual case of ALL in which ETV6 is found fused to the ABL gene; ABL is normally activated by fusion to the BCR gene in the 9:22 translocation. We expanded the primary cells from this ETV6/ABL rearranged case of ALL in SCID animals and analyzed them for expression of both ETV6/ABL and the normal ETV6 mRNA. We found that both the rearranged and normal ETV6 mRNAs are expressed in the expanded cell population. Furthermore, sequence analysis of the ETV6 PCR product revealed no point mutations which would influence the amino acid sequence. Thus, deletion of the second ETV6 allele is not necessary for the transformation to leukemia by ETV6/ABL.

Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer.

Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996; Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.

Mutations at codon 974 of the DPYD gene are a rare event.

A mutation at codon 974 of the dihydropyrimidine dehydrogenase (DPD) gene was previously described in a cancer patient with undetectable DPD enzyme activity who experienced severe toxicity when treated with 5-fluorouracil. We have studied the frequency of this mutation in 29 Scottish subjects with low DPD enzyme activity and in 274 American subjects. We detected no mutations in the 606 alleles studied and conclude that mutations at codon 974 are a rare event.

Rapid intraclonal switch of lineage dominance in congenital leukaemia with a MLL gene rearrangement.

We describe a case of neonatal mixed lineage leukaemia which presented with a dominant B progenitor lymphoblast population plus a minor monocytic component. Treatment of the patient with corticosteroid and Ara-C resulted in loss of lymphoblasts and a rapid (within 7 days) increase and dominance of the monocytic component. The common clonal origin of the two cell types was evident from the identical rearrangement in the MLL gene and a shared rearrangement of one IGH allele. In common with other neonatal or infant ALL with MLL gene rearrangements, this leukaemia may have originated in a common B-monocytic lineage stem cell during foetal haemopoiesis. The observations further suggest that the therapeutic impact of the MLL gene rearrangement is to some extent dependent on the cellular context in which it is expressed.

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event.

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT-PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.

RAS and FMS mutations following cytotoxic therapy for childhood acute lymphoblastic leukaemia.

Patients who have received cytotoxic therapy for primary neoplastic disease are at an increased risk of developing secondary (therapy-related) acute myeloid leukaemia (AML) or myelodysplasia (MDS). RAS and FMS mutations have been observed in patients with AML and MDS. It has been suggested that the mutational status within these genes may be predictive of early secondary leukaemic disease. In this study we have screened 50 haematologically normal patients in complete remission from childhood acute lymphoblastic leukaemia (ALL) for activating point mutations in the RAS and FMS proto-oncogenes. Such patients may be considered at risk of therapy-related disease. Codons 12, 13 and 61 were screened in RAS and codon 969 in FMS using the polymerase chain reaction (PCR) followed by oligonucleotide hybridization (ONH). Three of the 50 patients (6%) were found to harbour N12 RAS mutations. One of these three patients (2%) had both a N12 RAS and FMS 969 mutation. Upon sequencing the RAS mutations, substitutions of serine, cysteine and aspartic acid for glycine were identified. The FMS 969 mutation was also confirmed, by sequencing, as a histidine substitution. RAS mutations were not detected in presentation samples indicating that these lesions have been somatically acquired presumably subsequent to cytotoxic therapy for the primary disease. Continued follow-up of these patients may indicate a role for these mutations in the development of secondary malignancies.

Intrauterine monoclonal origin of neonatal concordant acute lymphoblastic leukemia in monozygotic twins.

We report detailed immunological, cytogenetic and molecular evidence for complete identity of the leukemic cell populations in monozygotic female twins with concordant leukemia diagnosed at two months of age. Both infants had early pre-B acute lymphoblastic leukemia with the (11;19)(q23;p13) chromosomal translocation. A common clonal origin of leukemia in these infants was suggested by the finding of identical oligoclonal heavy chain immunoglobulin gene rearrangements. Leukemic cell DNA was examined for 11q23 rearrangements by Southern blotting and restriction fragments of identical size were found in the two cases, in contrast to the diversity of rearrangements observed in other unrelated and nontwinned control infants with t(11;19)(q23;p13). Similar restriction fragments were absent in blood mononuclear DNA from both parents, liver tissue from one twin and remission bone marrow of the other, indicating that the 11q23 rearrangement was acquired and not inherited as a chromosomal abnormality or polymorphism. These findings provide a definitive evidence for intrauterine single cell origin, with twin to twin transmission, of concordant leukemia in this infant twin pair.

The novel activation of ABL by fusion to an ets-related gene, TEL.

In human leukemia, activation of the ABL proto-oncogene locus on chromosome 9 most commonly occurs as a result of its fusion to the BCR locus on chromosome 22. The resulting chimeric protein displays an elevated tyrosine kinase activity. We have identified a novel activation of ABL which involves a gene located on chromosome 12, designated TEL. Like BCR, TEL is fused in-frame with ABL and produces a fusion protein with an elevated tyrosine kinase activity when assayed in an immune complex. The amino-terminal sequences of TEL encode a helix-loop-helix motif which may mediate dimerization.

Neonatal mixed lineage acute leukaemia.

We report here an uncommon case of neonatal acute leukaemia that presented concomitant with serological evidence of rubella infection. The clinical course was aggressive and the patient died 5 days after diagnosis from septicaemia. Leukaemic blasts had a mixed lineage immunophenotype co-expressing a constellation of B-lymphoid (CD19, cytCD22, TdT) and myeloid (CD13, CD33, CD14, anti-MPO) markers, as well as multiple adhesion molecules and markers associated with early lympho-myeloid progenitor cells (CD34, CD7, HLA-DR). A previously unrecorded discordant expression of different CD10 and CD34 epitopes was identified using different monoclonal antibodies. The karyotype was 46,XX t(4;11)(q21;q23) and molecular analysis confirmed rearrangement of the trithorax-related oncogene HRX at 11q23. There was a clonal biallelic rearrangement of the immunoglobulin heavy-chain gene. The features of this rare case have implications for possible aetiological events leading to leukaemia.

Chromosome 11q23 abnormalities in leukaemia.

Breakpoints on chromosome 11 at band q23 have been observed in patients with primary or secondary leukaemia. Recent data have shown that these breakpoints are clustered in a approximately 15kb region of a gene named HRX. This gene product has homology to the Drosophila trithorax gene product, which suggests it may play a role in regulating transcription control. Disruption of HRX as a result of chromosomal translocation is thought to contribute to the leukaemogenic process; this may occur in utero giving rise to infant acute leukaemia or may be induced by epipodophyllotoxic drugs resulting in secondary leukaemia. Translocations of 11q23 can involve a number of different partner chromosomes. The reciprocal genes on chromosomes 4q21, 9p22 and 19p13 have been recently cloned and are predicted to encode proline and serine rich proteins. Of particular interest is the high degree of homology observed between the genes on 9p22 and 19p13, which suggests that they too may have an important role to play in the generation of the leukaemic phenotype.

Lack of MDM2 amplification in human leukaemia.

While deletion or mutation of the p53 gene is one of the most common molecular alterations detected in a wide variety of tumours, it has been shown to occur in only a relatively small percentage of the leukaemia cases examined. However, it may be that other components of the p53 pathway are involved. Amplification of the MDM2 gene has recently been demonstrated in human sarcomas resulting in an increase in MDM2 protein levels. This protein can bind to p53 preventing the transactivation of p53 responsive genes, thus mimicking mutation or deletion of p53. We have investigated the prevalence of MDM2 amplification in human leukaemias. 101 leukaemia or lymphoma samples and nine cell lines were studied using Southern blotting. In no case was MDM2 amplification present. We conclude that MDM2 amplification is not a common event in human leukaemias.

In utero rearrangements in the trithorax-related oncogene in infant leukaemias.

The majority (approximately 75%) of infant acute leukaemias have a reciprocal translocation between chromosome 11q23 and one of several partner chromosomes. The gene at 11q23 (named MLL, ALL-1, HRX or HTRX-1; refs 2-6) has been cloned and shares homology with the Drosophila developmental gene trithorax. Rearrangements of this gene (called HRX here) occur in introns and cluster in a region of approximately 10 kb; individual patients have different breakpoints. Here we describe three pairs of infant twins with concordant leukaemia who each share unique (clonal) but non-constitutive HRX rearrangements in their leukaemic cells, providing evidence that the leukaemogenic event originates in utero and unequivocal support for the intra-placental 'metastasis' hypothesis for leukaemia concordance in twins.

Expression of the colony-stimulating factor 1 receptor in B lymphocytes.

The FMS proto-oncogene encodes for the colony-stimulating factor 1 receptor (CSF-1R), whose expression within the haematopoietic system has previously been thought to be restricted to cells of the mononuclear phagocyte lineage. We have studied the expression of the CSF-1R in peripheral blood mononuclear cells by indirect immunofluorescence and flow cytometry. FMS expression was detected on both monocytes and B lymphocytes from all samples analysed, including 14 haematologically normal individuals and 31 patients (23 in remission following cytotoxic therapy for lymphoma, six with B-cell chronic lymphocytic leukaemia and two with chronic myelomonocytic leukaemia). The level of FMS expression on B lymphocytes was lower than the level of expression detected on monocytes isolated from the same sample. FMS mRNA expression in B lymphocytes has been confirmed by a reverse transcription-polymerase chain reaction (RT-PCR)-based technique and Northern blot analysis. Thus, FMS may play a role in the normal function of B lymphocytes and, because of its potential oncogenic activity, may contribute to the pathogenesis of malignancies of this cell type.

Breakpoints at 11q23 in infant leukemias with the t(11;19)(q23;p13) are clustered.

We have analyzed a series of nine infant leukemias that carry a t(11;19)(q23;p13). They had the morphologic features of acute lymphoblastic leukemia (ALL) and expressed markers typical of B-cell progenitor ALL or pre-B ALL; one coexpressed myeloid markers in addition to lymphoid markers (biphenotypic). Two probes (P/S4 and 98.40) subcloned from a yeast artificial chromosome (YAC) known to span the breakpoint in the t(4;11) were used to investigate DNA isolated from the leukemic cells of these patients. A total of approximately 15 kb of genomic DNA in the vicinity of the probes was examined by conventional Southern blot analysis using a series of restriction enzymes. In eight of the nine cases, the breakpoint could be mapped to an approximately 10-kb BamHI fragment disclosed by hybridization to the P/S4 probe.

FMS mutations in myelodysplastic, leukemic, and normal subjects.

The FMS gene encodes the functional cell surface receptor for colony-stimulating factor 1, the macrophage- and monocyte-specific growth factor. Codons 969 and 301 have been identified as potentially involved in promoting the transforming activity of FMS. Mutations at codon 301 are believed to lead to neoplastic transformation by ligand independence and constitutive tyrosine kinase activity of the receptor. The tyrosine residue at codon 969 has been shown to be involved in a negative regulatory activity, which is disrupted by amino acid substitutions. This study reports on the frequency of point mutations at these codons, in vivo, in human myeloid malignancies and in normal subjects. We studied 110 patients [67 with myelodysplasia (MDS) and 48 with acute myeloblastic leukemia (AML)], 5 patients being studied at the MDS and the later AML stage of the disease. There was a total incidence of 12.7% (14/110) with mutations in codon 969 and 1.8% (2/110) with mutations in codon 301. Two patients had mutations in the AML stage of the disease but not in the preceding MDS and one had a mutation in the MDS stage but not upon transformation of AML. This is consistent with the somatic origin of these mutations. FMS mutations were most prevalent (20%) in chronic myelomonocytic leukemia and AML type M4 (23%), both of which are characterized by monocytic differentiation. One of 51 normal subjects had a constitutional codon 969 mutation, which may represent a marker for predisposition to myeloid malignancy.