Expression of the putative tumor suppressor gene gravin and beta actin in acute leukaemias: clinical importance and prognostic value by real-time quantitative PCR

A-kinase anchor protein 12 [AKAP12] is a scaffold protein that participates in mitotic regulation and other signaling processes and probably exerts tumour suppressor function. Acute leukemias are caused by genetic and epigenetic mechanisms involving tumour suppressor genes and oncogenes. Epigenetic regulation plays a key role in the pathogenesis of leukemia. Aberrant DNA methylation patterns are the most frequent molecular alterations detected in AML. Whereas the pathogenetic importance of these changes has begun to emerge, DNA methylation has thus far only played minor role as a biomarker in diagnosis, prognosis prediction and treatment control. The aim of this study was to determine the expression of tumor suppressor gene gravin which belongs to the A kinase anchoring protein family in acute leukaemia patients samples and controls and to explore its possible prognostic importance. In this study, real time quantitative PCR was used to determine gravin gene expression and beta Actin was used as control gene and expression levels were compared with prognostic factors. Gravin gene expression was found to be decreased in 100% of patients as compared with the control group and it was found that there is significant correlation between its expression and laboratory prognostic markers, prognosis and .treatment outcome of acute leukemia patients. Gravin gene expression was found to be decreased in acute leukemias and the degree of its decreased expression has been found to be correlated with poor prognosis

Vascular endothelial growth factor [VEGF-C] signaling through KDR [VEGFR-2] and FLT-4 [VEGFR-3] mediates leukemic cell proliferation and survival

VEGF, a key angiogenic molecule, is a multifunctional cytokine that acts both as a potent inducer of vascular permeability and as a specific endothelial cell mitogen. Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of tumor angiogenesis. Leukemic cells not only release VEGF but also express its receptors, resulting in the establishment of an autocrine loop that supports their migration and survival. VEGF-C may play an important role in the pathophysiology of hematopoietic malignancies by not only regulation of lymphangiogenesis, in vivo, but also by promotion of angiogenesis invasion of neoplastic cells into lymphatic vessels and enhancing lymphatic metastasis during tumor progression. although it is well established that growth in solid tumors is dependent on the formation of neovasculature, the role of angiogenesis in hematopoietic neoplasms has not been determined. The present study was undertaken to identify whether VEGF-C and its receptors VEGFR-2 [KDR] and VEGFR-3 [FLT-4] were expressed in patients with denovo acute leukemia by RT-PCR and to evaluate the relationship between their expression and clinical, laboratory findings and prognosis. Using reverse transcription polymerase chain reaction analysis [RT-PCR], 30 de novo acute leukemia patients [20 ALL patients and 10 AML patients] as well as 10 controls were tested for the expressions of VEGF-C, VEGFR-3 [FLT 4] and VEGFR-2 [KDR] genes. In the current study, VEGF-C, FLT-4 and KDR were detected in 10% of control samples. In ALL patients VEGF C was expressed in 65% of cases, FLT-4 in 70% of cases and KDR in 30% of cases. The expressions of VEGF-C, FLT-4 and KDR in ALL patients were associated with increased risk of leukemia [with OR 16.7 and 95% CI 1.7-160.4, OR 21.0 and 95% CI 2.2-204.6 and with OR 3.9 and 95% CI 0.4-37.6 respectively]. In AML patients, VEGF-C was expressed in 60% of cases, FLT-4 in 70% of cases and KDR in 40% of cases. The expression of VEGF-C, FLT-4 and KDR in AML patients was associated with increased risk of leukemia [with OR 13.5 and 95% CI 1.2-152.2, OR 21.0 and 95% CI 1.8-248.1 and OR 6.0 and 95% CI 0.5-67.7 respectively]. In the 6 followed-up ALL patients, 3 [50%] were in remission, three of them were VEGF-C negative, 2 were FLT4 positive and 1 was KDR positive. 2 of the 6 ALL patients [33.3%] were resistant to treatment, both were VEGF-C positive, FLT-4 positive and 1 was KDR positive. One of the 6 ALL patients [16.6% died during induction, this patient was VEGF-C positive and FLT-4 and KDR negative. In the 4 followed-up AML patients, 3 of them [75%] were in remission, 1 of the 3 was VEGF-C positive and 2 were FLT-4 positive. One of the four AML patients [25%] was resistant to treatment, this patient was VEGF-C, FLT-4 and KDR positive. The number of VEGF-C positive patients with no treatment failure was lower than the number of VEGF-C positive patients with treatment failure. Also, the risk of failed induction was found to be greater in VEGF-C positive patients than in VEGF-C negative patients, thus, the expression of VEGF-C and its receptors [FLT-4 and KDR] in ALL and AML patients was associated with increased risk of leukemia and unfavorable treatment outcome. VEGF-C and its receptors KDR [VEGFR-2] and FLT-4 [VEGFR-3] may play an important role in the pathophysiology of hematopoietic malignancies and may actually contribute to the development of leukemia. Also, owing to the importance of angiogenesis in tumor progression and the effects of VEGF-C, KDR and FLT-4 in chemotherapy-treated leukemias, inhibition of VEGF-C signaling represents an attractive cancer treatment

Aberrant p15 promotor methylation in acute leukaemia

The p15 gene is one of the tumour suppressorgenes located on chromosome 9p21. In acute leukemias alterations involving the p15 gene have been reported. Commonly, these alterations involve the c[p]g islands that are commonly aberrantly methylated and result in the transcriptional loss of this gene. To detect this aberrant methylation, we used methylation specific pcr which is a novel method of pcr that can rapidly assess the methylation status of virtually any c[p]g island. The study included 30 patients: 17 cases of the novo all and 13 cases of de novo aml. Aberrant p15 gene mehylation was detected in 47.1% of cases of all and in 69.2% of casaes of AML. There was no statistically significant difference between methylated and unmethylated cases regarding the clinical and haematological data other than the peripheral blood blast cell count. On following up the patients to detect the response to therapy, there was a statistically significant difference in the response to therapy between methylated and unmethylated cases [p< 0.05]. Methylated case had a higher incidence of relapse or death [in all methylated cases 35.4% of the studied cases relapsed and 61.6% in aml patients] while the inidence of remission was 11.7% for all methylated cases and 7.7% for aml cases. Unmethylated all cases achieved remission in 41.2% of the studied group and unmethylated cases of AML reached remission in 61.6%. Aberrant p15 methylation may have important prognostic implications for clinical monitoing. And risk assessment. Also it opens new strategies of treatment using demethylating agents that can reverse this epigenetic change

Granulocyte macrophage colony stimulating factor: role in induction of monocyte CD14 receptor and adhesion molecules expression in chronic liver diseases

This study included 36 chronic liver disease [CLD] patients who suffered from viral hepatitis and / or schistosomiasis and 12 age and sex matched healthy individuals who represent the control group. The study aimed at clarifying the role of rhGM-CSF on the release of sICAM-1 and sCD14 from PBMNC. According to the severity of liver disease, patients were classified to Child A, B and C groups. All patients and controls were subjected to thorough history taking and clinical examination, a full routine laboratory investigation including hemogram, liver function tests and hepatitis markers. PB mononuclear cell culture was performed to all the study groups with and without the addition of rhGM-CSF to the culture media. Afterwards, slCAM-1 and sCDl4 were measured in culture supernatant fluid using ELISA technique, Levels of sICAM-1 in culture supernatants with and without addition of rhGM-CSF showed significant progressive increase with advancement of CLD which may reflect the increase ofsICAM-1 in sera of CLD patients with progression of the disease. As well, the addition of rhGM-CSF to PBMNC culture resulted in a significant reduction of sICAM-1 level in culture supernatants in control and patients groups in comparison to its level without the addition of rhGM-CSF. There was a significant progressive increase in sCD14 level with the advancement of the disease. The increase in sCD14 level with and without addition of rhGM-CSF was significant in all patients groups in comparison to the control group. As well, the addition of rhGM-CSF to culture media led to significant reduction of sCD14 concentration in supernatants in control group and in each of the patients groups in comparison to their levels without the addition of rhGM-CSF. It can be concluded that rhGM-CSF might be considered as one of the potential future tools against defective monocyte functions in CLDs. Using rhGM-CSF to improve monocyte functions will be associated with reduction of sICAM-1 and sCD14 levels which might be implicated or contribute to liver pathology