Ekspresija gena ABCB1 i gena regulatora apoptoze kao faktora rezistencije na hemioterapiju kod obolelih od hronične limfocitne leukemije

Abstract

Hronična lifocitna leukemija (HLL) je maligno oboljenje hematopoetskog tkiva, koje se manifestuje kao klonska ekspanzija B limfocita. HLL se karakteriše izuzetno heterogenim kliničkim tokom i odgovorom na terapiju, u čemu genetički profil pacijenta ima ulogu. Glavnu prepreku uspehu terapiji koja se koristi u lečenju kancera je rezistencija na lekove (eng. Multi Drug Resistance, MDR). Rezistencija na lekove se različito manifestuje kod pacijenata i genetički je determinisana. Dva glavna mehanizma učestvuju u rezistenciji na lekove koji se primenjuju u lečenju kancera: povišena ekspresija proteina koji su efluksne pumpe i ne dozvoljavaju ulazak leka u ćeliju (transportna, pumpna rezistencija), i povišena aktivnost antiapoptotskog sistema (apoptotska rezistencija). Ovo je prva studija genetičkih markera transportne i apoptotske rezistencije na lekove u pacijenata sa HLL u Srbiji. Analizirana je ekspresija ABCB1 gena čiji je produkt protein ćelijske membrane koji učestvuje u efluksu lekova iz ćelija, kao i uticaj dve varijante u ABCB1 genu (rs1045642, c.3435C > T i rs20132582, c. 2677G > T/A), na njegovu ekspresiju. Pored toga izučavana je i ekspresija Bcl2 i Bax gena, koja može da ukaže na status apoptotskog sistema u ćelijama pacijenata obolelih od HLL. Ekspresija gena je izučavana u mononuklearnim ćelijama periferne krvi 46 HLL bolesnika i 53 zdrave kontrole metodom reverzne transkripcije i lančane reakcije polimeraze u realnom vremenu (qRT-PCR), dok je detekcija varijanti u ABCB1 genu urađena metodom lančane reakcije polimeraze i digestijom restrikcionim enzimom (PCR-RFLP). U našoj studiji nije detektovana statistički značajna razlika u učestalosti varijantnih alela u ABCB1 genu, c.3435C > T i c.2677G > T/A, između zdravih kontrola i pacijenata sa HLL. Ovaj podatak ukazuje na to da ove genetičke varijante nisu faktori rizika za oboljevanje od HLL. U našoj grupi HLL pacijenata, 40% pacijenata je imalo visoku ekspresiju ABCB1 gena, koja se značajno razlikuje od ekspresije ovog gena koja je slična novou ekspresije kod zdravih kontrola. Može se reći da je u 40% HLL pacijenata ekspresija ABCB1 gena pozitivan marker za potencijalnu pojavu rezistencije na lekove (MDR). Naši rezultati nisu pokazali asocijaciju varijanti ABCB1 gena, c.3435C > T i c.2677G > T/A, niti varijantnih genotipova, sa ekspresijom ABCB1 gena, slično mnogim studijama. Rezultati molekularno-genetičkih analiza su bili korelisani sa odgovorom na terapiju u HLL pacijenata. Marker terapijskog odgovora je bio postizanje kompletne ili parcijalne remisije.

Chronic lymphocytic leukemia (CLL) is a hematologicam malignancy which manifests as monoclonal expansion of B lymphocytes. CLL is characterized by extremely variable clinical presentation, with different therapy requirements and overall survival. Genetic profile of a patient can be used as a predictor of the course of the diseases and therapeutic response. A major impediment to the success of human cancer therapy is the development of cancer variants exhibiting multidrug resistance (MDR). MDR is characterized with various manifestations and it is associated with individual genetic profile of a patient. Two most important mechanisms contribute to MDR, including the overexpression of drug efflux pumps (transport, pump resistance) and the upregulation of cellular antiapoptotic defense systems (apoptotic resistance). This is the first study of genetic markers of transport and apoptotic MDR in CLL patients in Serbia. Expression оf ABCB1 gene, coding for a cell membrane protein which mediates the efflux of drugs, as well as the effect of two ABCB1 gene variants (rs1045642, c.3435C > T and rs20132582, c. 2677G > T/A) on its expression were analyzed. Furthermore, expression of Bcl2 and Bax genes, regulators of apoptosis in CLL cells, was studied. Gene expression was analyzed in perypheral blood mononuclear cells of 46 CLL patients and 53 controls, using qRT-PCR (Real-time PCR), while detection of ABCB1 gene variants was performed using PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) methodology. Frequency of ABCB1 variant allelles, c.3435C > T and c.2677G > T/A, did not show statistically significant difference between CLL patients and healthy controls. This finding suggests that analyzed variants are not risk factors for CLL. When compared to healthy controls, ABCB1 gene overexpression was detected in 40% of CLL patients. Consequently, expression of ABCB1 gene could be considered a positive marker of potential development of MDR in 40% of CLL patients. Similar to many other studies, our findings did not reveal the association of either ABCB1 gene variants, c.3435C > T и c.2677G > T/A, or variant genotypes, with ABCB1 gene expression. The results of molecular genetic analysis were correlated with therapy response of CLL patients. Achieving a complete or a partial remission was chosen to be a therapy response marker. Analysis of genetic markers of transport MRD in CLL patients revealed that elevated level of ABCB1gene expression was associated with partial, but not with complete remission. Our finding suggests that ABCB1gene expression could be considered as a potential marker of transport MDR in CLL patients treated with FC protocol. Analysis of genetic markers of apoptotic MRD in CLL patients revealed that elevated level of Bcl2/Bax ratio was associated with partial, but not with complete remission. Our finding suggests that Bcl2/Bax ratio could be considered as a potential marker of apoptotic MDR in CLL patients treated with FC protocol. This study contributes to knowledge on importance of analyzed genetic markers for chemotherapy response in CLL patients and for individualization of therapy for these patients.