Biomedical subjects

Juhani Vilpo

Publications and source records attributed to Juhani Vilpo.

At least 19 recordsLinked to original sources

PubMed2005

Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status.

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate this further, we analyzed the VH and VL gene rearrangements in 90 VH3-21+ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VH genes and 37% unmutated VH genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased Vlambda2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant lambda expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographic origin and mutation status.

Amino Acid Sequence↗

PubMed2005

Mitogen induced activation, proliferation and surface antigen expression patterns in unmutated and hypermutated chronic lymphocytic leukemia cells.

OBJECTIVES: To determine whether the immunoglobulin V(H) gene mutational status has an effect on the activation, proliferation and surface antigen expression of chronic lymphocytic leukemia (CLL) cells when stimulated in vitro. METHODS: The proliferation and activation responses of CLL cells were studied in 22-immunoglobulin gene V(H) unmutated (UM-CLL) and 12 hypermutated (M-CLL) CLL cases in 4-day cultures. As the mitogen responses have been previously shown to be diverse in CLL, a case-specific strategy based on optimized mitogen combinations (OMCs) of interleukin-2 (IL-2), 12-O-tetradecanoylphorbol 13-acetate (TPA), Staphylococcus aureus Cowan 1 (SAC), and human recombinant tumor necrosis factor alpha (TNF) was applied in cell stimulation. The expression of 23 surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD38, CD40, CD45, CD45RA, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, IgD, and IgM) was studied by flow cytometry at days 0 and 4. RESULTS: The proliferation and activation responses were similar in UM-CLL and M-CLL when OMCs contained IL-2, TPA or TNF. SAC induced faster proliferation in UM-CLL than in M-CLL. OMC stimulation induced preferential down-regulation of growth- promoting cell surface receptors CD5, CD21, and CD124 and preferential up-regulation of growth-inhibiting antigen CD80 in M-CLL. CONCLUSIONS: Difference in immunophenotypic evolution of UM-CLL and M-CLL can be demonstrated if appropriate matrix signals are provided. The pathways for CD5, CD21, CD124 (IL4R), and CD80 (B7-1) regulation should be further explored in relation with somatic hypermutation and outcome of CLL.

Antigens, CD↗

PubMed2005

Patients with chronic lymphocytic leukemia with mutated VH genes presenting with Binet stage B or C form a subgroup with a poor outcome.

BACKGROUND AND OBJECTIVES: The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. DESIGN AND METHODS: VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. RESULTS: After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. INTERPRETATION AND CONCLUSIONS: CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.

Adult↗

PubMed2004

Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia.

We recently identified a chronic lymphocytic leukemia (CLL) subgroup using the immunoglobulin variable heavy-chain (V(H)) gene V(H)3-21 with almost identical heavy-chain complementarity determining region 3s (HCDR3s) and preferential variable light-chain (V(L)) gene usage, suggesting recognition of a common antigen epitope in this subset. To further explore the B-cell receptors (BCRs) in CLL, we characterized 407 V(H) rearrangements amplified from 346 CLLs regarding V(H), diversity (D), and joining (J(H)) gene usage and performed multiple alignment of the HCDR3 sequences. These analyses revealed 3 small subsets (2 V(H)1-69 groups, 7 cases; and 1 V(H)1-2 group, 5 cases) with highly restricted HCDR3 features including identical V(H)/D/J(H) usage, HCDR3 lengths, and shared N-sequences, in addition to the V(H)3-21 group (22 cases). Furthermore, another 3 groups (9 V(H)1-3(+) cases, 3 V(H)1-18(+) cases, and 5 V(H)4-39(+) cases) had essentially identical V(H)/D/J(H) use and similar HCDR3 lengths but less conserved N-regions. Analysis in all 6 of these subgroups showed restriction in V(L) gene use, whereas no association between V(H) and V(L) usage was found in cases without HCDR3 similarities. Altogether, structurally similar HCDR3s associated with preferential V(L) gene usage implies selection of BCRs, especially in subsets showing high HCDR3 similarities, thus pointing to restricted antigen recognition sites and possibly involvement of specific antigens in CLL development.

Adult↗

PubMed2004

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia.

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.

Aged↗

PubMed2004

Novel adamantylated pyrimidines and their preliminary biological evaluations.

The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea, thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective bromo-, thio- and S-alkyl derivatives. The molecular structures for some compounds were studied by X-ray methods. The significant anticancer and antimicrobial properties of [2-(6-adamantan-1-yl-2-methylpyrimidin-4-ylthio)ethyl]dimethylamine were found.

Adamantane↗

PubMed2004

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 2.5 mM) was allowed to react with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC and HPLC-MS technique. Although the predominant reaction observed was chlorambucil hydrolysis, 1 reacted with various heteroatoms of the nucleosides. The principal site of alkylation with all pyrimidine nucleosides was N3, as judged by 1H NMR and HPLC-MS analyses. Also, several other adducts were detected, which could be tentatively characterized by means of HPLC-MS and MS/MS. As expected, thymidine was the least reactive pyrimidine nucleoside studied, and in addition of the N3 derivative, it reacted only at the carbohydrate moiety. Overall reactivity of cytosine nucleosides with 1 was considerably higher. The N3 adducts of dCyd and 5-Me-dCyd partially deaminated under the reaction conditions employed, but the reaction was not catalyzed by the participation of the omega-hydroxy function of the alkyl substituent but presumably by the nitrogen atom of the chlorambucil moiety. In the case of cytosine nucleosides, the O2 derivatives were the second most abundant species. 5-Me-dCyd reacted more readily at O2 than dCyd. These O2 adducts were labile under acidic, neutral, and basic conditions. No N4 derivatives or cross-links were detected, but dCyd reacted also at C5, although the yield of this derivative was very low. The role of chlorambucil-pyrimidine 2'-deoxyribonucleoside adducts on the cytotoxicity and mutagenity of 1 is also discussed.

Antineoplastic Agents, Alkylating↗

PubMed2004

Similar humoral immunity parameters in chronic lymphocytic leukemia patients independent of VH gene mutation status.

Chronic lymphocytic leukemia (CLL) is a clonal B-cell disorder, which has recently been divided into 2 subtypes based on the somatic hypermutation status of the immunoglobulin heavy chain (IgVH) genes. In patients with unmutated tumor cells the survival time is approximately half of that in mutated cases, but the reason for this difference is poorly understood. Since infections are the major cause of mortality in CLL, we investigated the effect of the mutation status on host immunity and proneness to infections in patients with CLL. As expected, the disease progression seemed to be faster and the disease more advanced (Binet B and C) among unmutated patients than in the mutated ones. Surprisingly, no differences in humoral immunity [immunoglobulin G (IgG), IgM, IgA, IgG subclasses, anti-ABO blood group antibodies and mannan-binding lectin (MBL)] or immune responses (Haemophilus influenzae serotype b conjugate vaccination) were detected between these 2 patient groups. Furthermore, UM-patients were not more prone to infections compared to M-patients, and therapy had no impact on the incidence and pattern of infections in either of the patient groups. The current findings within this patient cohort reveal that the worse outcome in the unmutated subgroup is not caused by more severe defects in immunity and increased susceptibility to infections when compared with the hypermutated group. It is thus conceivable that active immunization procedures such as vaccination can successfully be applied on patients with unmutated IgVH gene and advanced disease stage.

Aged↗

PubMed2003

Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vlambda2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope.

The immunoglobulin variable heavy chain (IgVH) gene mutation status is an important prognostic factor in chronic lymphocytic leukemia (CLL), since cases with mutated VH genes show significantly longer survival than unmutated cases. Recently, we reported a preferential use of the VH3-21 gene in mutated CLL and showed that mutated VH3-21 cases had an inferior overall survival compared with other mutated CLL. In order to further characterize this subset, we performed VH gene analysis in 265 CLL cases and identified 31 VH3-21 cases (11.7%); 21 cases had mutated and 10 cases unmutated VH genes. Regardless of VH gene mutation status, a poor overall survival was found in the VH3-21 cases with a median survival of 83 months. These survival data confirm that VH3-21 cases do not fit into the general prognostic grouping of mutated and unmutated CLL. A large fraction of VH3-21 cases also demonstrated unique features with shorter lengths of the third complementarity determining region (CDR3) and CDR3s with highly homologous amino acid sequences. Furthermore, the VH3-21 cases showed a striking dominance of lambda light chain expression, and analysis of the Iglambda gene rearrangements revealed highly restricted use of the Vlambda2-14/Jlambda3 genes in the majority of cases. Taken together, our new findings strengthen the suggestion that VH3-21-using cases comprise a new CLL entity, irrespective of VH gene mutation status, and implicate that a common antigen epitope, perhaps of pathogenic significance, is recognized by the highly homologous VH3-21/Vlambda2-14 Ig molecules expressed in individual tumors.

Amino Acid Sequence↗

PubMed2003

Induction of SOS response, cellular efflux and oxidative stress response genes by chlorambucil in DNA repair-deficient Escherichia coli cells (ada, ogt and mutS).

Chlorambucil (CLB) is a bifunctional alkylating drug widely used as an anticancer agent and as an immunosuppressant. It is known to be mutagenic, teratogenic and carcinogenic. The cellular actions of CLB have remained poorly investigated. It is very likely that DNA damage and its repair are the key elements determining the destiny of CLB-exposed cells. We investigated the role of two specific DNA repair pathways involved in CLB-induced mutagenicity and gene expression changes by using Escherichia coli strains lacking either (i) two DNA methyltransferase functions (O(6)-methylguanine-DNA methyltransferase I (ada) and II (ogt)), or (ii) mismatch repair (MutS (mutS)). Mutagenicity was determined as the development of ciproxin and rifampicin resistance and the gene expression changes were assessed using expression profiling of all E. coli 4290 open reading frames (ORFs) by cDNA array. Chlorambucil-induced mutants in mutS cells, implying the importance of mismatch repair in preventing CLB-induced mutations. It also induced mutants in the ada, ogt strain, but to a lesser extent than in the wild-type strain. The simultaneous upregulation of several genes of the SOS response, cellular efflux and oxidative stress response, was demonstrated in both of the DNA repair-deficient strains but not in the wild-type cells. These and our previous results show that single-gene knock-out cells use specific gene regulation strategies to avoid mutations and cell death induced by agents such as chlorambucil.

Adenosine Triphosphatases↗

PubMed2003

More extensive genetic alterations in unmutated than in hypermutated cases of chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is not a uniform disease entity; approximately half of the CLL cases have undergone immunoglobulin V(H) gene hypermutation, whereas the other half display unmutated V(H) genes. We investigated genome changes in 12 hypermutated cases (M-CLL) and 22 unmutated cases (UM-CLL) by use of comparative genomic hybridization, G-banding, and multicolor fluorescence in situ hybridization (m-FISH) after optimal mitogen stimulation and FISH analysis of typical CLL aberrations: 11q deletion, 13q deletion, and trisomy 12. Very high frequencies of aberrations were found in both groups: 82% in UM-CLL and 83% in M-CLL. Deletions of 11q and 13q were equally distributed in M-CLL and UM-CLL. However, larger aberrations detectable by CGH, trisomy 12, and complex aberrations were less frequent in M-CLL than in UM-CLL. These observations led to a hypothesis that unmutated and mutated CLL have different biological Backgrounds, given that large and/or complex chromosomal aberrations and hypermutation of the CLL progenitor cells tend to be mutually exclusive.

Chromosome Aberrations↗

PubMed2003

Ex vivo drug and irradiation sensitivities in hypermutated and unmutated forms of chronic lymphocytic leukemia cells.

Several investigators have now established that chronic lymphocytic leukemia (CLL) is not a uniform disease entity, since approximately half of the cases of CLL have undergone immunoglobulin V region (IgV) hypermutation, whereas the other half display unmutated Ig genes. The median survival time of mutated CLL (M-CLL) cases has been shown to be approximately twice as long as that for unmutated CLL (UM-CLL), but no clear explanation for this difference is currently available. In this work, we have investigated a cohort of previously untreated CLL patients, to see whether the ex vivo sensitivities of leukemic cells of 16 UM-CLL patients differ from those of 8 M-CLL patients, using nine different drugs and two types of irradiation. Our results demonstrated very similar ex vivo sensitivities and tumor cell heterogeneity of sensitivity of UM-CLL and M-CLL cells when tested against chlorambucil, 2-chloro-2'-deoxyadenosine, cyclosporin A, cis-platinum(II)diammine-dichloride, doxorubicin hydrochloride, 2-fluoroadenine-9-beta-D-arabinofuranoside, prednisolone sodium succinate, verapamil, vincristine, gamma-irradiation, and UV-irradiation. This indicates that de novo chemo/radiosensitivity cannot explain the survival difference observed between UM-CLL and M-CLL.

Antineoplastic Agents↗

PubMed2003

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyadenosine.

N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 0.6 mM) was allowed to react with 2'-deoxyadenosine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC-MS and HPLC-MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 7% of 1 reacted with various heteroatoms of the nucleoside. The principal site of alkylation was N1. Several other adducts were also detected. The N1, N6, N3, and N7 derivatives were characterized by means of MS/MS, UV, and (1)H NMR. The N6 adduct is derived directly from alkylation of N6 of 2'-dAdo. Dimroth rearrangement of the N1 adduct to the N6 adduct was very slow under the reaction conditions employed. Minor adducts such as a carbohydrate derivative were tentatively characterized by MS/MS. No cross-links were detected. The role of chlorambucil-2'-deoxyadenosine adducts in the cytotoxicity and mutagenicity of 1 is also discussed.

Antineoplastic Agents, Alkylating↗

PubMed2003

Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia.

Recent studies have demonstrated that B-cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy-chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM-CLL and M-CLL patients. The similarity of M-CLL and UM-CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogeneous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M-CLL and UM-CLL. More positive cells in the UM-CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M-CLL cases, but only 36% of UM-CLL cases, were Ig-lambda+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M-CLL, whereas the GMF intensity of CD45RA tended to be associated with UM-CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.

Antigens, Surface↗

PubMed2003

Abnormal expression of apoptosis-related genes in haematological malignancies: overexpression of MYC is poor prognostic sign in mantle cell lymphoma.

The expression of apoptosis-related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real-time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33-positive ALL, and in CD56-positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P < 0.01). AML patients with a normal karyotype showed a higher frequency of BCL2A1 overexpression (P < 0.001) than those with an abnormal karyotype.

Adult↗

PubMed2003

Vaccination against infections in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a well-defined mature B-cell neoplasm associated with increased susceptibility to infections. Two major options in prevention of infections in CLL, intravenous gammaglobulin treatment and antimicrobial chemoprophylaxis, have not resulted in satisfactory outcome. A third strategy, antimicrobial vaccination, is the topic of this minireview. We collected articles and their references concerning CLL vaccination from the Medline database starting from 1966 and thirteen relevant studies were found. Plain bacterial polysaccharide vaccines would seem to be ineffective in antibody formation in patients with CLL. However, protein and conjugate vaccines appear to be more immunogenic and their responses may be further enhanced with ranitidine adjuvant treatment. New well-designed investigations are needed to develop appropriate vaccination strategies and evaluate vaccination efficacy in infection morbidity and mortality in CLL.

Bacterial Vaccines↗

PubMed2003

Lymphotoxin beta expression is high in chronic lymphocytic leukemia but low in small lymphocytic lymphoma: a quantitative real-time reverse transcriptase polymerase chain reaction analysis.

BACKGROUND AND OBJECTIVES: The lymphotoxin beta (LTB) gene, localized to the major histocompatibility complex region on chromosome 6p21.3, has an important role in the formation of germinal center reactions and regulation of immune response and apoptosis. Our aim was to determine LTB gene expression in different hematologic neoplasias. DESIGN AND METHODS: We determined the expression of LTB using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on a series of RNA samples from CD3(+) T cells and CD19(+) B cells isolated from peripheral blood (n=7); CD19(+) B cells isolated from lymph nodes (n=11) and from patients with acute lymphoblastic leukemia (ALL; n=16), acute myeloid leukemia (AML; n=43), chronic myeloid leukemia (CML; n=12), mantle cell lymphoma (MCL; n=19), chronic lymphocytic leukemia (CLL; n=32) and small lymphocytic lymphoma (SLL; n=22). RESULTS: The expression level of LTB in CD3(+) T cells and CD19(+) B cells isolated from blood was ten to forty times lower than that in normal CD19(+) B cells isolated from lymph nodes. In malignant myeloid cells the expression levels were very low, whereas in malignant lymphoid cells the expression was higher than in myeloid cells, being highest in MCL and CLL (20.2+/-14.0 ng/microL and 81.0+/-116.3 ng/microL) and low in SLL (4.5+/-3.6 ng/microL; p=0.001). We did not find correlations between LTB expression and hematoclinical parameters (risk groups, immunophenotypes and overall survival). INTERPRETATION AND CONCLUSIONS: A distinct difference in expression of LTB in CLL and SLL indicates that these morphologically similar B-cell malignancies are molecularly different. Further studies are needed to investigate the prognostic value of LTB and any role that LTB may have in determining whether the malignant B cells manifest a leukemia or lymphoma.

Adult↗

PubMed2002

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain.

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.

Alkylating Agents↗