Ekspresija gena ABCB1 i gena regulatora apoptoze kao faktora rezistencije na hemioterapiju kod obolelih od hronične limfocitne leukemije
Expression of ABCB1 gene and apoptotic regulatory genes as markers of multidrug resistance developed to chemotherapy used for the treatment of chronic lymphocytic leukemia.
Doktorand
Vučićević, Ksenija V.Mentor
Pavlović, SonjaČlanovi komisije
Đurđević, PredragStamatović, Dragana
Jakovljević, Vladimir
Metapodaci
Prikaz svih podataka o disertacijiSažetak
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 w...hich 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.