Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes.

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband's fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.

Congenital lactic acidosis, cerebral cysts and pulmonary hypertension in an infant with FOXRED1 related complex 1 deficiency.

Mitochondrial complex I is encoded by 38 nuclear-encoded and 7 mitochondrial-encoded genes. FOXRED1 is one of the 13 additional nuclear genes known as assembly factors. So far, four patients have been described with complex I deficiency caused by autosomal recessive mutations in FOXRED1. Here, we report the fifth patient with FOXRED1 related complex 1 deficiency presenting with prenatal onset of bilateral periventricular cysts, congenital lactic acidosis, and persistent life-limiting pulmonary hypertension. Whole exome sequencing identified a compound heterozygosity for a known pathogenic variant (c.612_615dupAGTG; p.A206SfsX15) (paternal) and a likely pathogenic variant (c.874G>A; p.Gly292Arg) (maternal). Deficiency of complex I was demonstrated by the absence of complex I on Blue Native Gel Electrophoresis and by a significantly reduced complex I enzyme activity in the patient's fibroblasts. Compared with the previous known FOXRED1 cases, unique clinical features observed in our patient include bilateral periventricular cysts and severe pulmonary hypertension. Whole exome sequencing was instrumental in recognizing the underlying gene defect in this patient.

Congenital lactic acidosis, cerebral cysts and pulmonary hypertension in an infant with FOXRED1 related complex I deficiency.

Mitochondrial complex I is encoded by 38 nuclear-encoded and 7 mitochondrial-encoded genes. FOXRED1 is one of the 13 additional nuclear genes known as assembly factors. So far, four patients have been described with complex I deficiency caused by autosomal recessive mutations in FOXRED1. Here, we report the fifth patient with FOXRED1 related complex 1 deficiency presenting with prenatal onset of bilateral periventricular cysts, congenital lactic acidosis, and persistent life-limiting pulmonary hypertension. Whole exome sequencing identified a compound heterozygosity for a known pathogenic variant (c.612_615dupAGTG; p.A206SfsX15) (paternal) and a likely pathogenic variant (c.874G > A; p.Gly292Arg) (maternal). Deficiency of complex I was demonstrated by the absence of complex I on Blue Native Gel Electrophoresis and by a significantly reduced complex I enzyme activity in the patient's fibroblasts. Compared with the previous known FOXRED1 cases, unique clinical features observed in our patient include bilateral periventricular cysts and severe pulmonary hypertension. Whole exome sequencing was instrumental in recognizing the underlying gene defect in this patient.

Mitochondrial DNA Deletions With Low-Level Heteroplasmy in Adult-Onset Myopathy.

We report the cases of 2 patients who presented to our Myositis Center with myalgias and elevated creatine kinase levels. Muscle biopsy showed pathological features consistent with mitochondrial myopathy. In both cases, a single large deletion in mitochondrial DNA at low-level heteroplasmy was identified by next-generation sequencing in muscle tissue. In 1 case, the deletion was identified in muscle tissue but not blood. In both cases, the deletion was only identified on next-generation sequencing of muscle mitochondrial DNA and missed on array comparative genome hybridization testing. These cases demonstrate that next-generation sequencing of mitochondrial DNA in muscle tissue is the most sensitive method of molecular diagnosis for mitochondrial myopathy due to mitochondrial DNA deletions.

Whole exome sequencing reveals de novo pathogenic variants in KAT6A as a cause of a neurodevelopmental disorder.

Neurodevelopmental disorders (NDD) are common, with 1-3% of general population being affected, but the etiology is unknown in most individuals. Clinical whole-exome sequencing (WES) has proven to be a powerful tool for the identification of pathogenic variants leading to Mendelian disorders, among which NDD represent a significant percentage. Performing WES with a trio-approach has proven to be extremely effective in identifying de novo pathogenic variants as a common cause of NDD. Here we report six unrelated individuals with a common phenotype consisting of NDD with severe speech delay, hypotonia, and facial dysmorphism. These patients underwent WES with a trio approach and de novo heterozygous predicted pathogenic novel variants in the KAT6A gene were identified. The KAT6A gene encodes a histone acetyltransfrease protein and it has long been known for its structural involvement in acute myeloid leukemia; however, it has not previously been associated with any congenital disorder. In animal models the KAT6A ortholog is involved in transcriptional regulation during development. Given the similar findings in animal models and our patient's phenotypes, we hypothesize that KAT6A could play a role in development of the brain, face, and heart in humans. © 2016 Wiley Periodicals, Inc.

Mitochondrial DNA content varies with pathological characteristics of breast cancer.

Changes in mitochondrial DNA (mtDNA) content in cancers have been reported with controversial results, probably due to small sample size and variable pathological conditions. In this study, mtDNA content in 302 breast tumor/surrounding normal tissue pairs were evaluated and correlated with the clinico-pathological characteristics of tumors. Overall, mtDNA content in tumor tissues is significantly lower than that in the surrounding normal tissues, P < 0.00001. MtDNA content in tumor tissues decreased with increasing tumor size. However, when the tumor is very large (>50 cm(3)), mtDNA content started to increase. Similarly, mtDNA content decreased from grades 0 and I to grade II tumors, but increased from grade II to grade III tumors. Tumors with somatic mtDNA alterations in coding region have significantly higher mtDNA content than tumors without somatic mtDNA alterations (P < 0.001). Tumors with somatic mtDNA alterations in the D-Loop region have significantly lower mtDNA content (P < 0.001). Patients with both low and high mtDNA content in tumor tissue have significantly higher hazard of death than patients with median levels of mtDNA content. mtDNA content in tumor tissues change with tumor size, grade, and ER/PR status; significant deviation from the median level of mtDNA content is associated with poor survival.

Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids.

Somatic mitochondrial DNA alterations have been found in all types of cancer. To better understand the role of mitochondria and their involvement in the pathogenic mechanisms of cancer development, the effects of cancer mitochondria were investigated in a defined nuclear background using a transmitochondrial cybrid system. Our results demonstrated that cancer mitochondria confer a significant reduction in cell growth when cells are metabolically stressed in a galactose medium. Activities of the respiratory chain complexes, cellular oxygen consumption, and ATP synthesis rates were found to be much lower in breast cancer cells, than those in normal breast epithelial cells of MCF-10A (10A). These results suggest that there is reduced mitochondrial function in the studied breast cancer cell lines. Similarly reduced mitochondrial function was observed in cybrids containing cancer mitochondria. Novel tRNA mutations were also identified in two breast cancer cell lines, possibly responsible for the observed mitochondrial dysfunction. We conclude that altered mitochondria in cancer cells may play a crucial role in tumor development.

Mitochondrial DNA variant interactions modify breast cancer risk.

Interactions between mitochondrial deoxyribonucleic acid (mtDNA) variants and the risk of developing breast cancer were investigated using DNA samples collected from non-Jewish European American breast cancer patients and ethnically age-matched female controls. Logistic regression was used to evaluate two-way interactions between 17 mtDNA variants. To control for multiple testing, empirical P values were calculated using permutation. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to measure the contribution of variants in modifying the risk of developing breast cancer. A highly significant interaction was identified between variants 12308G and 10398G (empirical P value = 0.0028), with results suggesting these variants increase the risk of a woman developing breast cancer (OR = 3.03; 95% CI 1.53-6.11). Nominal significant P values were also observed for interactions between mtDNA variants 709A and 16189C; 4216C and 10398G; 4216C and 16189C; 10398G and 16159C; 13368A and 16189C; and 14766T and 16519C. However, after adjusting for multiple testing, the P values did not remain significant. Although it is important to elucidate the main effect of mtDNA variants on the risk of developing breast cancer, understanding gene x gene interactions will give a greater knowledge of disease etiology and aid in interpreting a woman's risk of developing breast cancer.

Mitochondrial genetic background modifies breast cancer risk.

Inefficient mitochondrial electron transport chain (ETC) function has been implicated in the vicious cycle of reactive oxygen species (ROS) production that may predispose an individual to late onset diseases, such as diabetes, hypertension, and cancer. Mitochondrial DNA (mtDNA) variations may affect the efficiency of ETC and ROS production, thus contributing to cancer risk. To test this hypothesis, we genotyped 69 mtDNA variations in 156 unrelated European-American females with familial breast cancer and 260 age-matched European-American female controls. Fisher's exact test was done for each single-nucleotide polymorphism (SNP)/haplogroup and the P values were adjusted for multiple testing using permutation. Odds ratio (OR) and its 95% confidence interval (95% CI) were calculated using the Sheehe correction. Among the 69 variations, 29 were detected in the study subjects. Three SNPs, G9055A (OR, 3.03; 95% CI, 1.63-5.63; P = 0.0004, adjusted P = 0.0057), A10398G (OR, 1.79; 95% CI, 1.14-2.81; P = 0.01, adjusted P = 0.19), and T16519C (OR, 1.98; 95% CI, 1.25-3.12; P = 0.0030, adjusted P = 0.0366), were found to increase breast cancer risk; whereas T3197C (OR, 0.31; 95% CI, 0.13-0.75; P = 0.0043, adjusted P = 0.0526) and G13708A (OR, 0.47; 95% CI, 0.24-0.92; P = 0.022, adjusted P = 0.267) were found to decrease breast cancer risk. Overall, individuals classified as haplogroup K show a significant increase in the risk of developing breast cancer (OR, 3.03; 95% CI, 1.63-5.63; P = 0.0004, adjusted P = 0.0057), whereas individuals bearing haplogroup U have a significant decrease in breast cancer risk (OR, 0.37; 95% CI, 0.19-0.73; P = 0.0023, adjusted P = 0.03). Our results suggest that mitochondrial genetic background plays a role in modifying an individual's risk to breast cancer.

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness.

Patients with mitochondrial disease usually manifest multisystemic dysfunction with a broad clinical spectrum. When the tests for common mitochondrial DNA (mtDNA) point mutations are negative and the mtDNA defects are still hypothesized, it is necessary to screen the entire mitochondrial genome for unknown mutations in order to confirm the diagnosis. We report an 8-year-old girl who had a long history of ragged-red fiber myopathy, short stature, and deafness, who ultimately developed renal failure and fatal cardiac dysfunction. Respiratory chain enzyme analysis on muscle biopsy revealed deficiency in complexes I, II/III, and IV. Whole mitochondrial genome sequencing analysis was performed. Three novel changes: homoplasmic 15458T > C and 15519T > C in cytochrome b, and a near homoplasmic 5783G > A in tRNA(cys), were found in the proband in various tissues. Her mother and asymptomatic sibling also carry the two homoplasmic mutations and the heteroplasmic 5783G > A mutation in blood, hair follicles, and buccal cells, at lower percentage. The 5783G > A mutation occurs at the T arm of tRNA(cys), resulting in the disruption of the stem structure, which may reduce the stability of the tRNA. 15458T > C changes an amino acid serine to proline at a conserved alpha-helix, which may force the helix to bend. These two mutations may have pathogenic significance. This case emphasizes the importance of pursuing more extensive mutational analysis of mtDNA in the absence of common mtDNA point mutations or large deletions, when there is a high suspicion of a mitochondrial disorder.

Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer.

BACKGROUND: The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. METHODS: The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma) of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE), followed by direct DNA sequencing to identify the mutations. RESULTS: Fourteen somatic mtDNA mutations were identified in 55% (11/20) of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64%) were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. CONCLUSION: Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations.

3-phosphoinositide-dependent protein kinase-1 (PDK1) promotes invasion and activation of matrix metalloproteinases.

BACKGROUND: Metastasis is a major cause of morbidity and mortality in breast cancer with tumor cell invasion playing a crucial role in the metastatic process. PDK1 is a key molecule that couples PI3K to cell proliferation and survival signals in response to growth factor receptor activation, and is oncogenic when expressed in mouse mammary epithelial cells. We now present evidence showing that PDK1-expressing cells exhibit enhanced anchorage-dependent and -independent cell growth and are highly invasive when grown on Matrigel. These properties correlate with induction of MMP-2 activity, increased MT1-MMP expression and a unique gene expression profile. METHODS: Invasion assays in Matrigel, MMP-2 zymogram analysis, gene microarray analysis and mammary isografts were used to characterize the invasive and proliferative function of cells expressing PDK1. Tissue microarray analysis of human breast cancers was used to measure PDK1 expression in invasive tumors by IHC. RESULTS: Enhanced invasion on Matrigel in PDK1-expressing cells was accompanied by increased MMP-2 activity resulting from stabilization against proteasomal degradation. Increased MMP-2 activity was accompanied by elevated levels of MT1-MMP, which is involved in generating active MMP-2. Gene microarray analysis identified increased expression of the ECM-associated genes decorin and type I procollagen, whose gene products are substrates of MT1-MMP. Mammary fat pad isografts of PDK1-expressing cells produced invasive adenocarcinomas. Tissue microarray analysis of human invasive breast cancer indicated that PDK1pSer241 was strongly expressed in 90% of samples. CONCLUSION: These results indicate that PDK1 serves as an important effector of mammary epithelial cell growth and invasion in the transformed phenotype. PDK1 mediates its effect in part by MT1-MMP induction, which in turn activates MMP-2 and modulates the ECM proteins decorin and collagen. The presence of increased PDK1 expression in the majority of invasive breast cancers suggests its importance in the metastatic process.

Characterization of medroxyprogesterone and DMBA-induced multilineage mammary tumors by gene expression profiling.

Mouse mammary tumors arising during medroxyprogesterone-DMBA-mediated mammary carcinogenesis comprised three distinct phenotypes: adenocarcinoma, squamous cell carcinoma, and myoepithelial carcinoma. The molecular signature for each of the three tumor subsets was characterized by gene microarray analysis, and three distinct sets of gene expression profiles were obtained that were corroborated in part by quantitative RT-PCR and immunohistochemistry. These results suggest that this carcinogenesis and gene expression model will be useful for rapidly assessing the histopathological differences arising in mammary carcinogenesis and the effects of tumor promoting or chemoprevention agents.

Peroxisome proliferator-activated receptor delta and gamma agonists differentially alter tumor differentiation and progression during mammary carcinogenesis.

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid beta-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARgamma and PPARdelta agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARgamma agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARdelta agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARgamma agonist expressed estrogen receptor-alpha in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARdelta agonist exhibited increased PPARdelta levels and activated 3-phosphoinositide-dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARdelta activation. These results indicate that PPARdelta and PPARgamma agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.

[Restoring beta1 integrin activation function in K562 cells transfected with antisense VEGF121 cDNA].

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.

A cystic fibrosis patient with two novel mutations in mitochondrial DNA: mild disease led to delayed diagnosis of both disorders.

A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for unknown mutations in the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE). Her asymptomatic mother's blood DNA was also analyzed and used as a reference. Two tRNA regions showing different TTGE patterns between the proband and her mother were sequenced. Two novel mutations, G15995A in tRNA(pro) and A8326G in tRNA(lys), were revealed. These mutations are present in heteroplasmic states. They both occurred at a nucleotide position that is highly conserved throughout evolution. This patient is also a compound heterozygote for the cystic fibrosis (CF) mutations, DeltaF508 and R347P. The phenotype for R347P has been associated with mild disease. Due to the mild features of the R347P mutation in the CF transmembrane conductance regulator (CFTR) gene and the heterogeneous clinical presentation of the mtDNA disease, the patient was not definitively diagnosed until age 21. This case underscores the importance of a complete mutational analysis of the entire mitochondrial genome when a patient suspected of mitochondrial disorder is negative for common mtDNA mutations.

Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer.

To investigate the role of mitochondrial DNA (mtDNA) in tumorigenesis, a temporal temperature gradient gel electrophoresis method was used to analyze the somatic mtDNA mutations in breast cancer. The entire mitochondrial genomes in 19 tumor samples and paired normal tissues from the same patients were amplified using 32 pairs of overlapping primers. DNA fragments showing different banding patterns between normal and tumor mtDNA were sequenced to identify the mutations. Fourteen of the 19 tumors (74%) displayed at least one somatic mtDNA mutation. Twenty-seven somatic mutations were found, and 22 of them occurred in the D loop region. This study represents the most comprehensive mtDNA mutational analysis in breast cancer.

[Low Concentrations of STI571 Enhances beta1 Integrin Mediated Inhibitory Effect on Proliferation of Myeloid Progenitors in Ph(+)Chronic Myeloid Leukemia]

To investigate whether ABL specific tyrosine kinase specific inhibitor STI571 can restore beta1 integrin mediated negative effect on Ph(+) chronic myeloid leukemia(CML), the inhibitory effect of beta 1 integrin activator (beta1 integrin activating antibody 8A2, cytokines such as GM-CSF, G-CSF and SCF) and/or FN on the granulocyte-macrophage colony forming unit (CFU-GM) from 16 patients with Ph(+)CML and 13 normal individuals were examined; the bone marrow mononuclear cells (BMMNC) before and after ABL kinase specific inhibitor STI571 pretreatment (0.1 micro mol/L for 30-60 minutes) were target cells in this study. The roles which VLA4 and VLA5 played in this process were evaluated through blocking assay. The results showed: (1) beta1 integrin activator(s) or FN alone have no effect on CFU-GM from CML or normal bone marrow mononuclear cells before or after STI571 pretreatment, nor STI571 pretreatment itself. (2) The inhibitory effect of beta1 integrin activator(s) plus FN on CML CFU-GM are significantly lower than that on normal CFU-GM. (3) The inhibitory effect of beta1 integrin activator(s) plus FN on CML CFU-GM after STI571 pretreatment is comparable to that on normal CFU-GM. (4) Monoclonal antibody to VLA4 and VLA5 or to total beta1 integrins almost completely abrogate the above effect of STI571. The results suggested enhancing beta1 integrin mediated negative effect on myeloid progenitors in Ph(+)CML is one of the therapeutic mechanisms of STI571 on Ph(+)CML.

[Expression of Vascular Endothelial Growth Factor in the Bone Marrow Cells from Adult Chronic Myelogenous Leukemia]

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines and plays an important role in the development of human solid tumors. However, it is not clarified whether VEGF governs the progress of the chronic myelogenous leukemia (CML). This study is to estimate VEGF expression in the bone marrow cells from normal and adult CML patients and various leukemic cell lines. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of VEGF mRNA. VEGF concentrations in the cell cultural supernatant and the plasma from normal and CML patient bone marrows were determined by enzyme linked immunosorbent assay (ELISA). VEGF mRNA was positive in 67 of 72 cases of bcr/abl(+) CML patient bone marrow cells (93.1%), in 5 of 10 CML patients post Allo-BMT bone marrow cells (50%), and in 6 of 10 normal bone marrow cells (60%), the expression rate of VEGF mRNA in CML patients bone marrow cells was higher than that in CML patients post Allo-BMT and normal bone marrow cells. VEGF mRNA also expressed in the HL-60, K562, CEM, KG1a, NB4, and Nalm6 cells, but not in the Jurkat cells. The mean VEGF concentration in the plasma (380.6 pg/ml) from 22 untreated CML patients was 9 folds higher than that from 9 CML patients post Allo-BMT (38.0 pg/ml). The mean VEGF concentration in the cultural supernatant (499.8 pg/ml) of 17 newly diagnosed CML bone marrows was 2.5-folds higher than that in 11 normal donors (141.3 pg/ml). The CML marrow cells secrete more VEGF than normal marrow cells do. Our results suggest that the abnormality of VEGF transcription and translation expression may play an important role in the development of CML.

[beta1 Integrin Dysfunction in Adult Chronic Myeloid Leukemia Bone Marrow Cells]

According to our previous experiments, Ph(+) chronic myeloid leukemia (CML) cell line K562 cells have defects in beta 1 integrin activation. In order to search the same regularity in Ph(+) CML bone marrow cells, bone marrow mononuclear cells (BMMNC) from 12 cases of Ph(+) CML and 10 cases of normal individuals were studied. Their expression rate of 9EG7 epitope on beta1 integrin post treatment by 8A2 or GM- or G-CSF and cell adhesion ability with soluble fibronectin (FN) were evaluated by flow cytometry; in addition, the effects of CGP57148B, a highly specific ABL tyrosine kinase inhibitor, were observed. Our results showed that 9EG7 expression rate and FN binding rate were very low in all the inactivated cells. The parameter increased markedly post 8A2 activation in both NBMMNCs and CMLBMMNCs, but the degree of increase in CMLBMMNCs was significantly lower than that in NBMMNCs; GM-CSF or G-CSF could significantly increase the parameters in NBMMNCs while had no effects on that in CMLBMMNCs. CGP57148B could increase the beta1 integrin activation potential of CMLBMMNCs but had no effects on that of NBMMNCs. The results indicate that decreased activation potential of beta1 integrin in CMLBMMNCs is the major cause of adhesion defects of Ph(+) CML cells; beta1 integrin functional insufficiency in CMLBMMNCs could not be directly reversed by ABL tyrosine kinase inhibitor CGP57148B.