The influence of drug formulations on the expression of MDM2 and NFkB1 mRNA in the melanoma cell lines

Introduction. Chemotherapy is an extremely ineffective and unsatisfactory means of treating malignant melanoma due to drug resistance, which is characteristic of this disease. A number of studies have shown that liposomal forms of anticancer drugs are able to overcome the multidrug resistance, but the mechanism by which this occurs is still remained to be elucidated. Aranoza (DNA-alkylating agent, a derivative of nitrosourea) has been approved for the treatment of patients with metastatic melanoma. Objective: to examine the influence of liposomal aranoza as well as the empty liposomes and “liophilisate for the preparation of solution for injections” (aranoza-lio) on the expression of mRNA of p53, MDM2, NFkB1, NFkB2, MyD88. Materials and methods. The study was performed with 10 melanoma cell lines, 4 of which carried the BRAF mutation. The level of p53, MDM2, NFkB1, NFkB2, MyD88 mRNA was investigated by quantitative polymerase chain reaction in real time. Results. Aranoza-lio increased slightly the expression of p53 mRNA in BRAF-mutated cells. We have observed also the increased expression of MDM2 mRNA (p = 0.0013). The expression of NFkB2, MyD88 mRNA did not change significantly as compared to control. Liposomal aranoza increased the expression of NFkB1 mRNA. Conclusion. Based on the data obtained we conclude that the liposomal aranoza triggers the mechanisms that contribute to sensitivity of cells toward anticancer drugs while aranoza-lio favored the enhancing of the expression of MDM2 mRNA and increase the resistance to chemotherapy.

PROGNOSTIC SIGNIFICANCE OF MDM2 GENE EXPRESSION IN CHILDHOOD NEUROBLASTOMA

Aim: To investigate the association of MDM2 expression at the mRNA levels in neuroblastoma with clinical features and unfavorable disease factors to determine the possibility of it usage as a prognostic marker of neuroblastoma. Materials and Methods: Total RNA and DNA were extracted from tumor tissue samples of total 91 neuroblastoma patients (mean age: 39.45 ± 4.81 months). MDM2 mRNA levels were detected with Q-PCR. TP53 gene deletion was detected with FISH method. MYCN amplification was detected with Q -PCR analysis in fresh tumor samples and FISH in FFPE samples. Results: We investigated the association of MDM2 mRNA expression with clinical outcome in neuroblastoma patients (n = 91). Kaplan — Meier curves showed a significant association of high MDM2 expression with poor event-free survival (p < 0.001). Clinical outcome of patients without MYCN amplification with low MDM2 expression was associated with better event-free survival than with high MDM2 expression (p < 0.001). Overexpression of MDM2 can be used as significant prognostic marker for patient stratification on risk groups and treatment optimization. Conclusion: Our results showed that the high expression of MDM2 at mRNA levels is an important factor of neuroblastoma prognosis. It may be a valuable additional molecular marker in guiding specific therapy in MYCN non-amplified NB patients without TP53 gene deletion.

Association of p53 mutations with progression-free survival (PFS) and overall survival (OS) in EGFR-mutated non-small cell lung cancer (NSCLC) patients (p) treated with erlotinib.

e18143 Background: Advanced NSCLC p with EGFR mutations have a median PFS of 14 months (m) and OS of 27 m when treated with erlotinib. In NSCLC cell lines, tyrosine kinase inhibitors (TKIs) induce p53 translocation from the cytoplasm to the nucleus and subsequent upregulation of Fas and caspase activation leading to apoptosis, but this mechanism was defective in p53-null cells. We tested whether TP53 mutations influence outcome to erlotinib in EGFR-mutated p. Expression levels of the p53 repressor MDM2 were also examined. Methods: We assessed p53 status in pretreatment paraffin-embedded tumor samples from 93 erlotinib-treated, EGFR-mutated advanced NSCLC p. Mutations in exons 5, 6, 7 and 8 were screened by High Resolution Melting analysis followed by sequencing of the amplified products with non-wild-type (wt) melt curves. All mutant samples were re-confirmed by standard PCR and sequencing. Expression levels of MDM2 mRNA were determined by quantitative RT-PCR. Results: Mutations in exons 5-8 of TP53 were detected in 26 of 93 p (28%). We found an unusually high frequency of in-frame and frameshift deletions (23% of mutations), indicating that the spectrum of p53 mutations might be different in EGFR-mutated NSCLC. Mutations were less frequent in p with ECOG PS >2 and more frequent in p with the T790M mutation. OS was 15 m in the 16 p with missense mutations 31 m in p with wt p53 and not reached in p with non-missense mutations (P=0.04). PFS was 9 m for 14 p with mutations in one of the p53 DNA binding motifs (DBMs), compared to 19 m for wt p and 27 m for p with non-DBM mutations. MDM2 mRNA levels were significantly lower in tumors with p53 mutations, especially in DBM mutations. In the case of wt p, high MDM2 expression correlated with longer PFS and OS in p with wt p53. Conclusions: TP53 mutations co-exist with EGFR mutations in a significant number of p; missense mutations correlate with shorter OS and mutations in DBMs correlate with shorter PFS. This finding paves the way for the possibility of combining erlotinib with a drug restoring p53 function in those p harboring certain types of mutations in the TP53 gene.

MDM2 Promoter SNP309 Is Associated with An Increased Susceptibility to Chronic Lymphocytic Leukemia and Correlates with MDM2 mRNA Expression In Chinese Population

Abstract 2424 Objective: A single nucleotide polymorphism (SNP) at position 309 in the promoter region of MDM2 leading to increased expression of MDM2 and attenuated function of p53 has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. Because inactivation of p53 by deletion and/or mutations also impacts on the clinical course of CLL, we assessed the role of the SNP309 genotype in CLL among Chinese populations. Methods: The MDM2 SNP309 genotypes in 166 CLL patients and 260 healthy controls were detected by the PCR-RFLP method, which all CLL samples was confirmed by direct DNA sequencing. We correlated the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS). In addition, the correlation of the MDM2 SNP309 genotype with the MDM2 mRNA expression level was evaluated by QPCR. Results: 1. The MDM2 T309G genotype frequencies were 22.9% (T/T), 48.2% (T/G), and 28.9% (G/G) among the cases, and 31.5% (T/T), 54.2% (T/G), and 14.2% (G/G) in the control subjects, and the difference was statistically significant (P=0.001). Logistic regression analyses revealed that the SNP309 G/G genotype instead of T/G heterozygote was associated with a significantly increased risk of CLL (adjusted OR = 2.8; 95% CI 1.57–4.98; P <0.001 for 309 G/G and adjusted OR = 1.22; 95% CI 0.76–1.96; P= 0.401 for 309 T/G, respectively), compared with the SNP309 T/T genotype. Age at onset of CLL was similar irrespective of MDM2 status. The median age at diagnosis for the different genotypes was 65 years for T/T and 62 years for T/G, 63 years for G/G (P>0.05). 2. In the entire cohort, no correlation was shown between the MDM2 SNP309 genotypes and Binet stage, IGHV mutational status, p53 mutation/deletion and no association existed between any particular MDM2 SNP309 genotype, OS and TFS. 3. The frequency of MDM2 mRNA expression in GG genotype was significantly higher than that in T/G (P=0.026) and T/T genotypes (P=0.003). Furthermore, patients with p53 aberrations (mutated and deleted) MDM2 expression were higher than SNP 309 T/G (P=0.046) and T/T genotypes (P=0.001), but were similar with G/G genotype with p53 wild-type (P=0.532), which prompted us to study the role of the polymorphism in p53 wild-type individuals. 4. In the p53 wild-type groups, we also confirmed MDM2 expression levels with SNP 309 G/G (P<0.001) and T/G (P=0.009) genotypes were higher than T/T genotypes. Moreover, survival analysis showed that the patients with SNP309 G/G (mean, 27.5 months; 95% CI, 21.2–33.9 months, P= 0.021) and T/G genotypes (mean, 48.7 months; 95% CI, 36.5–61.3 months, P= 0.045) both had significantly shorter TFS than SNP309 T/T genotype (mean, 97.2 months; 95% CI, 62.5–131.8 months). Conclusions: The results suggest that MDM2 309G polymorphisms contribute to the risk of developing CLL. The unfavorable SNP309 G/G genotype was associated with a gene-dosage-dependent increase of MDM2 expression. MDM2 SNP309 was found to be associated with TFS in p53 wild-type Chinese populations. Disclosures: No relevant conflicts of interest to declare.

MDM2 SNP 309 Is Not Associated with Increased Expression Levels of MDM2 Nor Clinical Significance in Childhood Acute Lymphoblastic Leukemia

Previously, we reported that MDM2 overexpression by pediatric acute lymphoblastic leukemia (ALL) cells is linked to a poor response to chemotherapy and early relapse of disease. It was recently shown that a single nucleotide polymorphism (SNP) in the MDM2 promoter, SNP309, was related to increased MDM2 expression and poor outcomes for various malignancies. The present retrospective study investigates the association of SNP 309 with MDM2 expression levels and clinical significance in pediatric ALL. Data obtained from 81 children with ALL showed a similar genotype distribution of SNP 309 as seen in other types of cancer and healthy populations: 37 T/T (46%), 37 T/G (46%) and 7 G/G (8%). However, this SNP did not seem to control MDM2 mRNA expression in ALL. We observed that 20 of the 81 ALL cases overexpressed MDM2 mRNA (&gt; 10-fold higher than in normal bone marrow mononuclear cells), of which 8 were SNP 309 T/T, while 12 were T/G (10) or G/G (2) (p&gt;0.5). Furthermore, we found that both MDM2 mRNA and protein were undetectable in 10 of 37 T/G and 3 of the 7 G/G cases. In addition, there were no associations found between SNP 309 and the in vitro response to doxorubicin, nor and early relapse in the 81 ALL patients. Upon review of 23 ALL cell lines, we found there was a relatively higher percentage of T/G (57%) than in fresh ALL samples (46%). However, the expression level of MDM2 was not related to SNP 309, but was rather closely associated with p53 status: All 11 ALL lines with wild-type p53 overexpressed MDM2 and all 12 ALL lines with mutant p53 or with a p53-null phenotype expressed no or low levels of MDM2, regardless of their SNP 309 status. These results suggest that in pediatric ALL, the SNP 309 status affects neither the expression of MDM2 nor the clinical prognosis.

Transcriptional Inactivation of p53, Bax, Bcl-2 and Mdm2 Correlates with Malignant Transformation of the Uterine Cervix

Deregulation of the apoptotic machinery plays a major role in cell death, cellular transformation and cancer. p53, Bcl-2, Bcl-XL, Bax and Mdm2 mRNA expression patterns were evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer compared to those of normal cervical tissues, and correlated with the underlying cervical lesions. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. p53, Bcl-2, Bax and Mdm2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (p=0.003, p=0.009, p=0.040 and p=0.001, respectively). Specifically, p53, Bax and Bcl-2 exhibited substantially lower transcript levels in CIN lesions compared to controls, whereas Bax mRNA levels showed a significant decrease in cancer compared to normal specimens. Mdm2 mRNA expression was considerably lower in cancer than in CIN lesions or normal cervix. High-grade squamous intraepithelial lesions exhibited lower p53 and Bcl-2 mRNA levels than controls (p=0.002, p=0.016). Coexpression analysis revealed more correlations between the above apoptosis-related molecules in normal tissues compared to CIN or cancer specimens. p53 showed significant coexpression with Bax, Bcl-2 and Mdm2 (p=0.040, p=0.013 and p=0.015, respectively) in normal cervical specimens. Bax and Bcl-XL mRNA expression was negatively correlated. Mdm2 transcriptional levels correlated significantly with those of Bax, Bcl-XL and Bcl-2. Our findings show that p53, Bax, Bcl-2 and Mdm2 mRNA expression levels correlate with the malignant transformation of the uterine cervix. mRNA coexpression patterns of the members of the pro- and anti-apoptotic family examined in cervical carcinogenesis were found to be disrupted in CIN and cancer, as already demonstrated at the protein level.