Juhani Vilpo

Mammalian DNA nucleotide excision repair reconstituted with purified protein components

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Synthesis, antiprotozoal and anticancer activity of substituted 2-trifluoromethyl- and 2-pentafluoroethylbenzimidazoles.

The synthesis of several halogenated benzimidazoles substituted in position 2 with trifluoromethyl, pentafluoroethyl and 2-thioethylaminodimethyl group is reported. Antiprotozoal and anticancer activity of series of newly synthesized and previously obtained compounds was studied. All of tested bezimidazoles showed remarkable antiprotozoal activity against Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. Of the studied collection of halogenated benzimidazoles the most anticancer-active was the 5,6-dichloro-2-pentafluoroethyl compound, particularly against breast and prostate cancer cell lines.

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

Summary. 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.

Distinct gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA,PBX3, EBI2, TCF1, CGRP, CD14, IL8,GZMK, GPR17 and CD79B, was associated (P < 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s).

Frequent loss of the 11q14-24 region in chronic lymphocytic leukemia: A study by comparative genomic hybridization

The genetic basis and molecular pathogenesis of chronic lymphocytic leukemia (CLL) and the molecular mechanisms responsible for its progression remain poorly understood. Here, karyotyping techniques specifically optimized for CLL, comparative genomic hybridization (CGH), and fluorescence in situ hybridization were used to search for CLL‐specific genetic aberrations. CGH and karyotyping both revealed copy number changes in 12 of the 25 CLL cases (48%) analyzed. Loss at 11q emerged as the most common aberration (6 cases), followed by a gain of chromosome 12 (4) and loss at 13q (3). Concordance between CGH and G‐banding was found in 23 of the 25 cases (92%), which is more than reported in a recent similar CGH study of CLL. Owing to the basic differences in G‐banding and CGH, however, their simultaneous clinical application is recommended. The frequent loss of 11q14‐24 suggests that this chromosomal region deserves further attention as a candidate locus involved in the pathogenesis of CLL. Genes Chromosom. Cancer 19:286–290, 1997.

Response to vaccination against different types of antigens in patients with chronic lymphocytic leukaemia

We investigated responses to vaccination against pneumococcal polysaccharide, Haemophilus influenzae b (Hib) conjugate and tetanus toxoid antigens in 31 patients with chronic lymphocytic leukaemia (CLL) and 25 controls. While in the control group all antibody responses against different antigens were highly significant, in the patient group clear evidence for responsiveness was detected only in the case of Hib polysaccharide antigen. Certain CLL patient subgroups showed low reactivity against tetanus toxoid antigen. In conclusion, plain polysaccharide vaccines seem to be ineffective in patients with CLL. Conjugate vaccines, in turn, are immunogenic and may offer protection against infections caused by encapsulated bacteria in these patients. Further studies concerning an optimal vaccination scheme and clinical efficiency are warranted.

UV- and γ-irradiation-induced DNA single-strand breaks and their repair in human blood granulocytes and lymphocytes

Ionizing irradiation and UV-irradiation cause DNA damage. Ionizing irradiation induces single-strand breaks, much less abundantly double-strand breaks, alkali-labile sites, and various oxidized purines and pyrimidines. UV-irradiation, on the other hand, causes cyclobutane pyrimidine dimers, (6-4) photoproducts, and various monomeric base damages. The deposition of energy in DNA may result directly in single-strand breaks (predominant form after ionizing radiation), or the strand breaks may be generated during the repair process (predominant form after UV-irradiation). We investigated the formation and repair of DNA single-strand breaks in human blood granulocytes and lymphocytes by the single-cell gel electrophoresis or comet assay. The induction and repair of DNA lesions by gamma-irradiation was comparable in human blood granulocytes and lymphocytes. The finding is consistent with the expression of the pertinent base excision repair proteins in these cells. In contrast to gamma-irradiation, fewer single-strand breaks were observed immediately after UV-irradiation; the maximum number of breaks were seen when the cells were incubated for 30-60 min. After an incubation period of 150 min, a significant reduction of single-strand breaks was noted. It is conceivable that the first 30-60 min represented a period during which the incision-excision phase of nucleotide excision repair (NER) predominated. After that, strand joining was dominant, evidently representing the synthesis and ligation phase of NER. These results indicate that the approx. 30 different polypeptides required for complete NER are functional in these mature blood cells. This is the first demonstration of the expression of global NER in human granulocytes.

Surface antigen expression in chronic lymphocytic leukemia: clustering analysis, interrelationships and effects of chromosomal abnormalities.

Chronic lymphocytic leukemia (CLL) is a phenotypically distinguishable form of B-lymphoid leukemias. The regularity of surface membrane antigen expression patterns, their interrelationships as well as the effects of the three frequent chromosomal aberrations, ie 11q deletion, 13q deletion and trisomy 12, were investigated in 35 classic CLL cases by flow cytometry. The two-way cluster analysis of 31 individual antigens revealed three expression patterns: (1) most cells in most cases positive (CD5, CD19, CD20, CD23, CD27, CD40, CD45, CD45RA); (2) most cells in most cases negative (CD10, CD14, CD34, CD122, CD154, mIgG); and (3) a mixed pattern with a variable number of positive cases and a variable percentage of positive cells in individual cases (CD11c, CD21, CD22, CD25, CD38, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, mIgD, mIgκ, mIgλ, mIgM). The expressions of several antigens were strongly interdependent, even when antigens belonged to entirely different gene families. Such antigen pairs were: CD11c/CD21; CD19/CD45; CD19/CD79b; CD22/CD45RA; CD23/Igκ; CD25/mIgM; CD27/CD45; CD45/CD79b; CD45RA/Igκ. In contrast, the expression of some antigens was mutually exclusive, the best examples being CD45RA/CD45RO, CD38/CD80 and CD45RA/CD80. Deletion of chromosome arm 11q attenuated expression of splicing variant CD45RA, but enhanced CD45RO expression. In contrast, cases of trisomy 12 were associated with enhanced CD45RA and attenuated CD45RO expression. Similarly, trisomy 12 was associated with enhanced CD27 and mIgκ expression. The variable levels of signaling surface membrane antigens, their interactions and interference by genetic aberrations are likely to affect the clinical progression and drug response of CLL.

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.

The G(-248)A polymorphism in the promoter region of the Bax gene does not correlate with prognostic markers or overall survival in chronic lymphocytic leukemia

The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.