Transcript Diapositiva 1 - Hospital Italiano de Buenos Aires

EXPRESSION OF MCL1, GSTP1, AND CKS1B GENES IN PATIENTS WITH
MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE AND MULTIPLE
MYELOMA
Stella F1, Fantl D2, Arbelbide J2, Schutz N2, Slavutsky I1.
1Instituto
de Medicina Experimental (IMEX) CONICET-Academia Nacional de Medicina, 2Servicio de Hematología, Hospital Italiano,
Buenos Aires, Argentina. [email protected]
Background
Materials and Methods
Monoclonal gammopathy of undetermined significance
(MGUS) and multiple myeloma (MM) comprise heterogeneous
plasma cell disorders with incompletely understood
molecular defects. Microrray studies have identified genes
differentially expressed in MM, some of them, like MCL1,
GSTP1 and CKS1B, associated to drug resistance.
MCL1 (myeloid cell leukemia sequence 1) is an anti-
apoptotic member of the Bcl-2 family which is tightly
regulated during myeloid and B-cell differentiation. Mcl-1
overexpression in myeloma cells mediates cell proliferation
and delays apoptosis, contributing to disease relapse
(Leukemia. 2005; 19:1248-52; Nature 2010; 463:103-107).
GSTP1 (glutathione S-transferase ) belongs to a family of
enzymes that play an important role in detoxification by
catalyzing the conjugation of many hydrophobic and
electrophilic compounds (Haematologica 2000; 85:573-579).
CKS1B (CDC28 protein kinase regulatory subunit 1B) is a
member of the highly conserved CkS1 protein family that
interacts with cyclin-dependent kinases and plays a critical
role in cell cycle progression. In MM cells, deregulation of
CKS1B was associated with an aggressive clinical course
(Oncotarget. 2010; 1: 22–33).
Population studied: Bone marrow samples were obtained from
90 patients with plasma cell disorders at diagnosis. All
individuals gave informed consent and the study was approved
by the local Ethics Committee. Age, sex and clinical
characteristics of patients are described in Table 1.
Table 1: Population studied
Characteristics
MM
MGUS
60
30
34/26
21/9
66.4 (30-86)
70.6 (41-84)
Cases, n
RNA extraction and cDNA syntesis: Total RNA was extracted
from either mononuclear cells of normal donors and bone
marrow samples from patients using the Trizol Reagent
(Invitrogen). cDNA was then synthesized from 1,5 g/l RNA
in a 20l reverse transcription reaction mixture using 50.000
U of RT- MMLV and random primers (Promega).
Sex (F/M)
B2M (mean; range), g/ml
1.29 (0.15-16.5) 0.30 (0.11-0.77)
Real Time PCR: Quantification of the complementary DNA
template was performed with real-time polymerase chain
reaction (QRT-PCR), using a Rotor-Gene Q (Quiagen),
Master Mix, Taq DNA polymerase, dNTP mix, Eva Green Dye
(Byodinamics), and 500 pmoles of each primer (Table 2).
The relative expression of MCL1, GSTP1 y CKS1B genes were
performed using the beta actin constitutive expressed gene.
LDH (mean; range), U/I
213.8 (82-1265) 148.3 (94-231)
Median age (range), years
DS Stage (%)
I
21
II
17
III
62
Albumin (mean; range), mg/dl
3.33 (1.7-4.6)
3.74 (3-4.4)
Creatinine (mean; range), mg/dl
1.75 (0,6-11,8) 0.90 (0.46-1.82)
Serum calcium (mean; range), g/dl
9.29 (6.8-14.6)
M protein (mean; range), g
2.99 (0.1-9.45) 0.63 (0.18-1.73)
9.15 (7.8-10.3)
Haemoglobin (mean; range), g/dl 10.73 (6.9-14.8) 12.47 (9.5-15.8)
BMI, %
Statistic Analysis: Groupwise comparison of the distribution
of clinical parameters was performed using a two-tailed
Student t test or chi-square test. In addition Mann-Whitney
test and Kendal coefficient were used. For all test, p< 0.05
was considered significant.
43.29 (5-100)
6.31 (0-9.5)
Litic bone lesions, %
46.2
0
Renal failure, %
13.5
0
F: female; M: male; DS: Durie & Salmon;B2M: beta2 microglobulin;
LDH: lactate dehidrogenase; BMI: bone marrow infiltration
Table 2: Primers used in QRT-PCR analysis
Primer
A number of studies have reported the expression patterns
of these genes in MM but they have been scarcely, or even
not at all explored, in MGUS patients.
Sequence
Beta actin
PF: 5´-ATGTTTGAGACCTTCAACACCCC-3´
PR: 5´-GCCATCTCTTGCTCGAAGTCCAG-3´
MCL1
PF: 5´-GGGAATTACTGCAAAAATGGAATA-3´
PR: 5´-ATGACCGGAGACAAACAGAAC-3´
GSTP1
PF: 5´-CTCCGCTGCAAATACATCTC-3´
PR: 5´-ACAATGAAGGTCTTGCCTCC-3´
AIM
a)
The aim of this study was to evaluate mRNA expression of
MCL1, GSTP1 and CKS1B genes in patient with MGUS and MM
in order to analyze their participation in the progression of
disease.
b)
CKS1B
c)
PF: 5´-CGATCATGTCGCACAAACA-3´
PR: 5´-GCCAGCTTCATTTCTTTGGT-3´
QRT-PCR analysis: a) MCL1; b) GSTP1 and c) CKS1B melting curves;
PF: Primer forward; PR: primer reverse.
Results
Expression levels (X±SE) in patients and controls
Controls
MM H
MGUS H
MCL1
GSTP1
CKS1B
4.127±1.136
0.049±0.007 1.74E-03±4.51E-04
24.69 ±11.05* 0.27±0.135 2.91E-02±1.08E-02*
0.15±0.025* 8.45E-03±1.98E-03*
MM H: High expression MM group; MGUS H: High expression MGUS group; significant
differences with respect to controls *p<0.01
a) Significant differences
between beta2
microglobulin levels in
MM patients with high
and low expression of
MCL1 (p=0.015); b)
Percentage of bone
marrow infiltration in MM
and MGUS patients with
high versus low mRNA
CKS1B expression
(p<0.04)
CKS1B EXPRESSION
p=0.015
MCL1 groups
a)
b)
p=0.0005
p=0.0012
a)
b)
c)
Gene expression groups in patients and controls: a) MCL1 ; b) GSTP1 and c) CKS1B. MM H: MM High expression group; MM L: MM
Low expression group; MGUS H: MGUS High expression group; MGUS L: MGUS Low expression group.
a)
p<0.04
B2M (g/ml)
Group
b)
a) Positive
correlation
between mRNA
CKS1B
expression and
percentage of
bone marrow
infiltration
(p=0.0012;
r2=0.1942)
and b) protein
M levels
(p=0.0005;
r2=0.2415).
Abridgement of Results
Analysis of mRNA expression showed differences among evaluated genes,
supporting the high heterogeneity of these entities.
Overexpression of GSTP1 gene was observed in 32% of MM and 33% of MGUS
patients. Although MGUS mRNA levels (0.05±0.01) were lower than those of MM
(0.12±0.05), no significant differences between them were observed. GSTP1 gene
expression did not show association with clinical parameters.
 MCL1 had overexpression only in MM patients (24.69±11.05) with significant
differences with respect to controls (4.13±1.14) (p<0.0001).
The analysis of CKS1B also showed abnormal expression in a similar percentage of
MM and MGUS patients (60% and 50%, respectively), with higher CKS1B expression
in MM (0.017±0.006) compared to MGUS patients (0.004±0.001) (p<0.04) and in
both entities with respect to controls (0.002±0.0004) (p<0.04).
In MM, the correlation with clinical characteristics showed a positive association
between B2microglobulin and MCL1 mRNA levels (p=0.015). Expression of CKS1B
was positively correlated with the percentage of bone marrow infiltration
(p=0.0012; r2=0.1942) and M protein levels (p=0.0005; r2=0.2415).
Conclusion
- To our knowledge, this is the first evaluation of GSTP1 mRNA
expression in MGUS patients.
- Our data showed deregulation of GSTP1 and CKS1B genes in this
disorder.
- Significant differences in CKS1B expression between MM and MGUS
and a positive association with bone marrow infiltration suggest a
role for this gene in the progression from MGUS to MM.
- This results are in concordance with the only one report studying
CKS1B expression in MGUS patients (Brit J Haematol 2006; 134:
613-5), and probably reflect a more genetically unstable disease.