Abstract
Poly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA (ssDNA) breaks, thus protecting the cell from genomic instability. This enzyme is a promising target in anticancer treatment. The aim of the present study is to investigate the role of PARP1 in B chronic lymphocytic leukemia (B-CLL) patients pre- and post- treatment.
Blood samples were collected from B-CLL patients before treatment and following 3 cycles of immunochemotherapy (chosen by the treating physician according to common clinical practice). Quantification of PARP1-mRNA levels was performed by a SYBR-green based PCR performed on BIORADCFX96 (BIORAD). The results were expressed in correlation to the mRNA levels of the endogenous housekeeping gene of beta actin (ACTB). The detection of the gene product was performed by Western Blot using anti-PARP1 antibody (clone 47-258; Calbiochem). We detected the 70 KD protein segment that results from the in vivo fractionation of PARP1 by caspase-3. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated by densitometry using the gel analysis software ImageJ. We correlated the PARP1 protein levels to those of a group of healthy volunteers. We also studied the differences between PARP1-mRNA and protein levels before and after treatment and correlated them to clinical response. The related samples Wilcoxon Signed Rank test (IBM SPSS statistics, version 19.0) was used for the statistical analysis of the results.
Samples were obtained from 24 B-CLL patients before treatment and from 15/24 following 3 cycles of immunochemotherapy. The patients' characteristics are shown in Table 1. Pre-treatment PARP1 protein levels were similar to those of 15 healthy volunteers (p=0.426). No statistically significant difference was noted between the PARP1-mRNA and protein levels following treatment (p=0.507 and 0.650 respectively) (Table 1). Multivariate analysis did not reveal statistically significant differences in the mRNA and protein levels in correlation to the stage of disease, the peripheral blood lymphocyte count, the LDH levels, the response to treatment and the overall prognosis of the patients.
Patients' characteristics and results
| Characteristic . | Finding . | . |
|---|---|---|
| No of Subjects (pre/post-treatment) | 24 / 15 | |
| Age, median (range) | 74 (51-87) | |
| Male to female ratio | 1.5 | |
| Blood lymphocytes x109/L, median (range) | 29.4 (3.9-81.0) | |
| LDH/ULN1, median (range) | 1.2 (0.85-3.1) | |
| Previous treatment, N (%) | 2 (13.3) | |
| Stage (Binet) (15/24 patients), Ν (%) | ||
| A | 0 (0) | |
| B | 8 (53.3) | |
| C | 7 (46.7) | |
| Immunochemotherapy regimen, Ν (%) | ||
| R | 8 (53.3) | |
| RCh | 3 (20.0) | |
| FCR | 4 (26.7) | |
| Response after 3 cycles of treatment, Ν (%) | ||
| Complete Response (CR) | 5 (33.3) | |
| Partial Response (PR) | 8 (53.3) | |
| Stable Disease (SD) | 2 (13.3) | |
| Progressive Disease (PD) | 0 (0) | p |
| Pre-treatment PARP1-mRNA, median (range) | 0.094 (0.001 – 3.490) | 0.507 |
| Post-treatment PARP1-mRNA, median (range) | 0.055 (0.003 – 0.535) | |
| Pre-treatment protein, median (range) | 0.978 (0.789 – 1.201) | 0.650 |
| Post-treatment protein, median (range) | 1.057 (0.346 – 1.140) | |
| Protein, Control group (15 subjects), median (range) | 1.029 (0.572 – 1.344) | 0.4263 |
| Characteristic . | Finding . | . |
|---|---|---|
| No of Subjects (pre/post-treatment) | 24 / 15 | |
| Age, median (range) | 74 (51-87) | |
| Male to female ratio | 1.5 | |
| Blood lymphocytes x109/L, median (range) | 29.4 (3.9-81.0) | |
| LDH/ULN1, median (range) | 1.2 (0.85-3.1) | |
| Previous treatment, N (%) | 2 (13.3) | |
| Stage (Binet) (15/24 patients), Ν (%) | ||
| A | 0 (0) | |
| B | 8 (53.3) | |
| C | 7 (46.7) | |
| Immunochemotherapy regimen, Ν (%) | ||
| R | 8 (53.3) | |
| RCh | 3 (20.0) | |
| FCR | 4 (26.7) | |
| Response after 3 cycles of treatment, Ν (%) | ||
| Complete Response (CR) | 5 (33.3) | |
| Partial Response (PR) | 8 (53.3) | |
| Stable Disease (SD) | 2 (13.3) | |
| Progressive Disease (PD) | 0 (0) | p |
| Pre-treatment PARP1-mRNA, median (range) | 0.094 (0.001 – 3.490) | 0.507 |
| Post-treatment PARP1-mRNA, median (range) | 0.055 (0.003 – 0.535) | |
| Pre-treatment protein, median (range) | 0.978 (0.789 – 1.201) | 0.650 |
| Post-treatment protein, median (range) | 1.057 (0.346 – 1.140) | |
| Protein, Control group (15 subjects), median (range) | 1.029 (0.572 – 1.344) | 0.4263 |
1 ULN: Upper Limit of Normal
2 R, Rituximab; Ch, Chlorambucil; F, Fludarabine
3 p, correlation of pre-treatment protein levels of BCLL in patients and control group
The expression of PARP1 in patients with B-CLL seems to be comparable to that of healthy persons. We could not distinguish patients displaying reduced levels of PARP1, as previously reported by others. Moreover, PARP1 expression is not affected by immunochemotherapy, irrespective of stage and response to treatment. Thus, this molecule seems not to participate in the mechanisms involved in DNA repair and apoptosis that are impaired in B-CLL. The study is still ongoing with the addition of more subjects.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
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