Characteristics of novel myeloid precursor cell line, PC-MDS, established from a bone marrow of the patient with therapy-related myelodysplastic syndrome.

We report on characteristics of the first human cell line, PC-MDS, derived from a bone marrow of a patient with therapy-related myelodysplastic syndrome (t-MDS) who had no overt post-MDS leukemia. Classic cytology analyses, immunophenotyping, cytogenetic and molecular genetic procedures were used for characterization of the cell line. PC-MDS cells are positive for the expression of CD13, CD15, CD30, CD33, and CD45 antigen. Positive cytochemical staining and immunophenotype analyses indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell. The karyotype analysis of PC-MDS cell line revealed various numerical and structural changes including those typically associated with t-MDS: del(5)(q13)[7], der(5)t(5;11)(p11;q11)[13], -7[6], del(7)(q31)[2], +20[3], -20[4]. Evaluation of methylation status in a promoter region of p15, p16 and MGMT genes showed biallelic hypermethylation pattern of 5' promoter region only in MGMT gene. PC-MDS is the first t-MDS derived cell line, and based on its immunological, cytogenetic and molecular characterization could be a new tool in evaluation of complex biology of MDS and a model for methylation studies.

Comethylation of p16 and MGMT genes in colorectal carcinoma: correlation with clinicopathological features and prognostic value.

AIM: To investigate the significance of p16 and O(6)-methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. METHODS: p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. RESULTS: p16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCs, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation. Only 27% of patients with simultaneous p16 and MGMT methylation showed the detectable occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P < 0.05, chi(2)-test). In addition, p16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 +/- 6.0 mo vs 23.1 +/- 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P < 0.001, chi(2)-test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 +/- 1.9 mo vs 37.0 +/- 1.8 mo, P < 0.001, Log-rank test). The comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. CONCLUSION: Our data suggest that comethylation of promoters of p16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.

A Ph-negative chronic myeloid leukemia with a complex BCR/ABL rearrangement and a t(6;9)(p21;q34.1).

Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoetic stem cell characterized by the presence of the Philadelphia (Ph) chromosome in more than 90% of patients. Cryptic or "masked" BCR/ABL gene rearrangements may be found in cases with a normal karyotype and in cases with the complex karyotype, in which typical t(9;22) is not visible at the microscopic level. Those rearrangements can now be detected by fluorescence in situ hybridization. Here, we report on a novel and complex Ph chromosome-negative CML case with a t(6;9)(p21;q34.1) in which the BCR/ABL fusion gene is located at 6p21.

Elevated plasma TGF-beta1 levels correlate with decreased survival of metastatic breast cancer patients.

BACKGROUND: The role of circulating TGF-beta(1) in prognosis of breast cancer (BC) was investigated with an intention to define TGF-beta(1)-dependent high risk and low risk subsets of patients. METHODS: Fifty three BC patients of all clinical stages and 37 healthy donors (HD) were analyzed for plasma TGF-beta(1) by the TbetaRII receptor-based Quantikine TGF-beta(1) ELISA kit. RESULTS: The plasma TGF-beta(1) level of Stage I/II disease (median: 0.94 ng/ml; n=10)) remained close to HD (median: 1.30 ng/ml; n=37; p>0.1). In contrast, Stage III/IV disease (median: 2.34 ng/ml; n=43) exhibited highly significant TGF-beta(1) elevation (p<0.001) relative to HD. Further analysis revealed that TGF-beta(1) increase was predominantly attributed to Stage IV, metastatic disease patients (Q3=4.23 ng/ml) rather than to the group Stage III/IV (Q3=3.58 ng/ml). Using the plasma TGF-beta(1) concentration of 3.00 ng/ml as the cut-off value, two subgroups of patients were formed. Overall 2-year survival of the first subgroup, having elevated plasma TGF-beta(1) (>3.00 ng/ml; n=10), was 10%. This was significantly decreased (p<0.05) compared to 52% survival observed for the second subgroup of patients with plasma TGFbeta(1) values close to HD (<3.00 ng/ml, n=19). CONCLUSION: We have performed a pilot study to determine the relationship between overall survival and TGF-beta(1) concentration in the blood of metastatic breast cancer patients. The survival was significantly reduced in the patients with elevated plasma TGF-beta(1) levels compared to that of the patients with plasma TGF-beta(1) levels close to normal. We propose that plasma TGF-beta(1) concentration may be a new tumour marker attributed to the presence of metastatic BC cells that may be used in selection of metastatic BC patients with poor prognosis.

Hypermethylation of p15 gene in diffuse - large B-cell lymphoma: association with less aggressiveness of the disease.

In this study, methylation-specific polymerase chain reaction was used to investigate the potential prognostic significance of the methylation status of p15, p16, MGMT, and DAPK genes in 51 specimens of diffuse large B-cell lymphoma (DLBCL). Hypermethylation of p15 gene was significantly more prevalent in patients without relapse (p = 0.001) and there was a trend toward more frequent presence of p15 methylation in patients without death outcome within 5-year follow-up period (p = 0.086) Also, there was a trend toward accumulation of p15 methylation with favorable clinicopathological parameters including: age ≤ 60 years (p = 0.091), normal levels of lactate dehydrogenase (p = 0.090), Eastern Cooperative Oncology Group performance status < 2 (p = 0.095), and low/intermediate low International Prognostic Index (p = 0.076). In the female group and group of the patients without bulky tumor mass, treated with chemotherapeutic regimens including rituximab, methylation of p15 was significantly related to longer overall survival (p = 0.036 and 0.027, respectively). Our results suggest that promoter methylation of p15 gene could have prognostic value in DLBCL patients treated with rituximab when used in combination with gender and tumor size.

Prognostic significance of epigenetic inactivation of p16, p15, MGMT and DAPK genes in follicular lymphoma.

In this study, methylation-specific polymerase chain reaction was used to investigate the role and potential prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes in 32 specimens of follicular lymphoma (FL). Hypermethylation of p15 gene was associated with lower hemoglobin level (P = 0.020) and MGMT/DAPK comethylation with relapsed disease (P = 0.018). Among all patients with FL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated of any examined genes. Therefore, we analyzed methylation in the different groups according to FL International Prognostic Index (FLIPI) and tumor grade. In the high-risk group, patients with hypermethylated p16 gene had significant lower overall survival than those with unmethylated p16 (P = 0.006) and trend toward shorter failure-free survival (P = 0.068). In the same risk group, there was a trend toward longer overall survival for patients with hypermethylated MGMT gene, compared to those with unmethylated MGMT gene (P = 0.066). p15 methylation had impact on shorter overall survival in grade I group of patients (P = 0.013), and DAPK methylation tended to have impact on shorter failure-free survival in the whole examined group (P = 0.079). Our results suggest that promoter methylation of p16 and MGMT genes could have prognostic value when used in combination with the FLIPI and p15 methylation in combination with tumor grade. Concurrent methylation of MGMT and DAPK genes could be the marker of tumor chemoresistance and disease recurrence.

Concomitant aberrant methylation of p15 and MGMT genes in acute myeloid leukemia: association with a particular immunophenotype of blast cells.

In this study, methylation-specific polymerase chain reaction (MS-PCR) was used to define the methylation status of the target promoter sequences of p15 and MGMT genes in the group of 21 adult patients with acute myeloid leukemia (AML). The incidence of aberrant hypermethylation of p15 gene (71 %) was higher comparing to MGMT gene (33 %), whereas concomitant methylation of both genes had 24 % of the patients. Although the incidence of cytogenetic abnormalities between the groups with a different methylation status of p15 and/or MGMT genes was not significantly different, we observed general trend of clustering of abnormalities with adverse prognosis into groups with concomitant hypermethylation of both genes and only p15 gene. Also, we showed that AML patients with concomitant methylation of p15/MGMT genes had a higher proportion of leukemic blast cells characterized with specific expression of individual leukocyte surface antigens (CD117(+)/CD7(+)/CD34(+)/CD15(-)), indicating leukemic cells as early myeloid progenitors. Although we could not prove that hypermethylation of p15 and/or MGMT genes is predictive parameter for response to therapy and overall survival, we noticed that AML patients with comethylated p15/MGMT genes or methylated p15 gene exhibited a higher frequency of early death, lower frequency of complete remissions as well as a trend for shorter overall survival. Assessing of the methylation status of p15 and MGMT genes may allow stratification of patients with AML into distinct groups with potentially different prognosis.

Acute-phase protein gene expression in rat liver following whole body X-irradiation or partial hepatectomy.

We studied the effect of total body X-irradiation and partial hepatectomy on the acute phase protein gene expression in rat liver. Male rats of AO strain were irradiated with high X-ray doses, without any visible tissue damage. In contrast, partial hepatectomy consisted of surgical removal of 40% liver tissue. The changes in liver mRNA concentrations for positive acute-phase reactants including cysteine protease inhibitor, alpha(1)-acid glycoprotein, fibrinogen and haptoglobin, and albumin as a negative reactant were monitored by Northern blot and slot-blot hybridizations using corresponding [32P]dCTP labeled cDNA probes. While in the first 24 h after the partial hepatectomy, liver mRNA levels for the positive acute-phase reactants increased, briefly followed by an immediate decrease, the duration and timing of the acute-phase responses to the whole body X-irradiation were slightly different and lasted for as long as 72 h. Although both treatments induced the mRNA expression of acute-phase reactants in rat liver, the observed variations in the duration and intensity of the changes in mRNA levels for the acute-phase proteins in these two types of tissue damage suggest the involvement of specific mechanisms in a fine tuning of the non-specific acute-phase responses to meet the unique requirements of the particular injury.

Aflatoxin B1-induced changes of glutathione-S-transferase activity in the plasma and liver of the rat.

BACKGROUND: The influence of low doses of aflatoxin B1(AFB1) and partial hepatectomy (PH) on glutathione-S-transferase (GST) activity was studied in the plasma and liver of the rat. METHODS: The animals were divided into four groups. The first (I) and the second (II) group were treated with AFB1 freshly dissolved in dimethyl sulphoxide (DMSO), and administered as a single intraperitoneal dose of 50 micrograms/rat 24 hrs after the rats had undergone either sham operation or, 40% PH, respectively. The third group (III) of animals was treated with a total dose of 1 mg AFB1-5 days per week during a period of 8 weeks. The non-treated animals were used as controls (C). RESULTS: We observed a significant increase of GST activity in the plasma of all experimental groups compared to the controls (C), (p < 0.02 to p < 0.005). In the liver, the GST activity of all experimental groups was also significantly increased, compared to the controls (from p < 0.02 to p < 0.005). CONCLUSION: The administration of both single and multiple doses of AFB1 led to long term increase of GST activity in the rat plasma and liver, and partial hepatectomy had no significant effect on this phenomenon.