Targeting Bcl-2 in CLL.

Chronic lymphocytic leukemia (CLL) is a common adult leukemia in the Western world with an incidence of 4.2/100,000/year. The clinical course of disease is highly heterogenous; it affects people over 65-70 years of age. This hematologic cancer is characterized by the resistance to apoptosis stimuli predominantly associated with overexpression of antiapoptotic Bcl-2 family members. Therapeutic options for advanced CLL patients are limited. Thus there is an urgent need to discover a novel, less toxic, and much more effective agent(s) or drug combinations for CLL patients. Among chemotherapeutic(s) and immunotherapeutics currently in use, several enzyme inhibitors were tested to gain better results in CLL treatment. Here, we review the main achievements made on targeting of prosurvival Bcl-2 proteins through the use of different approaches, i.e. anti-sense methodology, small molecules that mimic the action of BH3 domain and microRNAs (mainly miRNA-15a and miRNA-16-1).

Enzyme-linked immunosorbent assay in screening of leukemia-associated nuclear proteins.

Our previous data revealed some diversities in electrophoretic characteristics of nuclear fraction proteins isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Two electrophoretically-specific nuclear non-histones in the molecular mass zone of 38/39 and 44/46 kDa of leukemic mononuclear cells were used as immunogens to produce rabbit antisera. The Western blot analysis indicated that both nuclear components are expressed only in mononuclear cells isolated from peripheral blood of B-CLL patients, but not in those isolated from the blood of healthy donors. For further investigations of nuclear fraction from normal and B-CLL mononuclear cells, an enzyme-linked immunosorbent assay (ELISA) was used. The results obtained by ELISA with the antisera raised against both electrophoretically-specific B-CLL nuclear polypeptides revealed a different extend of cross-reactivity of nuclear fraction preparations isolated from normal cells and those isolated from leukemic ones. We noticed that nuclear fraction preparations which originated from leukemic mononuclear cells are much more reactive than normal ones with both antisera (at a broad range of antisera dilutions).

Nuclear non-histone protein p36 associated with colorectal tumours.

Rabbit serum raised against electrophoretically specific nuclear polypeptides with molecular weights of 35-40 kD from colon adenocarcinoma has been used to detect p36 antigen in 83.3% (40 of 48) of cases of large intestine tumours by means of Western blot technique. Immunological analysis revealed that this antiserum cross-reacted with antigen of the same molecular weight in 83.3% (10 of 12) and 85.7% (6 of 7) nuclear protein preparations from stomach and lung tumours, respectively, but not in any control tissue samples. No cross-reactivity within the region of 36 kD was observed among nuclear proteins isolated from mononuclear cells of B-cell chronic lymphocytic leukaemia patients as well as healthy donors.

Cellular protein alterations in colorectal cancer: p28, p53 and p65 studies.

The expression and intracellular distribution of the p28 protein (MW 28 kD), which is electrophoretically specific for tumour cells, the p53 protein (MW 53 kD), one of the most frequently mutated in cancer, and the oncofoetal p65 protein (MW 65 kD), were investigated in colorectal cancer and normal colonic mucosa. The correlation between the expression of these proteins and the stage of the cancer, was evaluated. Neoplastic and normal tissues were fractionated by differential centrifugation, and protein analysis was performed by means of the Western blot technique in the presence of polyclonal (anti-p28 and anti-p65) or monoclonal (anti-p53) antibodies. Among the colorectal cancer cases examined 69% (11/16), 53% (10/19) and 77% (17/22) were positive for p28, p53 and p65, respectively. Immunoblot analysis revealed that the tumour specific p28 protein expression was mainly evident in the nuclear fraction, while the p53 and p65 proteins accumulated in the cell nuclei and the cytoplasm, although to different extents. The p65 protein appeared to be specifically expressed in the early stages of colorectal cancer, while a high level of p53 protein was typical for more invasive colorectal cancer stages.

Two-dimensional gel electrophoresis of nuclear proteins of different stages of B-chronic lymphocytic leukaemia.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the composition of nuclear proteins from normal and B-chronic lymphocytic leukaemia (B-CLL) mononuclear cells. Some differences in the electrophoretic behaviour of these proteins from normal and transformed cells, especially with molecular weights/pI of 14-16 kD/5.9-7.4; 28-32 kD/4.9-5.5; 38-39 kD/5.4-6.1; 44-46 kD/5.1-5.6; 47-52 kD/5.0-5.6; 64-69 kD/5.1-5.7, and 95-105 kD/5.2-5.5, were observed. The comparative analysis of nuclear proteins, obtained from mononuclear cells of patients with B-CLL at different stages of development, indicated that the expression of some protein components might be correlated with the progression of this disease.

Colorectal cancer-associated nuclear antigen.

By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with M(r) of 35-40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix.

A novel B-CLL specific nuclear protein (p44/46).

Our previous results indicated some diversities in electrophoretic patterns of proteins from different cellular fractions, i.e. nuclear, mitochondrial, microsomal and cytosolic isolated from mononuclear cells from the peripheral blood of B cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Major differences were observed in electrophoretic banding of nuclear proteins from normal and transformed cells, especially in molecular mass region of 37 52 kDa. Electrophoretically-specific nuclear protein with molecular mass of 44/46 kDa of cells originating from B-CLL patients was used for raising polyclonal antiserum. As it was determined by Western blot technique (with alkaline phosphatase) obtained antiserum recognized 44/46 kDa antigen of nuclear fraction from B-CLL and acute lymphoblastic leukemia (ALL) cells, but not from normal ones. Our preliminary data were revealed that this antiserum shows no crossreactivity with leukemic nuclear proteins of patients with T cell chronic lymphocytic leukemia (T-CLL) and neither with nuclear polypeptides from either normal or cancerous (adenocarcinoma) stomach and colon mucosa. Immunological analysis was shown that higher expression of this particular antigen seems to correlate with progression of B-CLL.

Molecular characterization of cellular proteins from colorectal tumors.

AIMS AND BACKGROUND: Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. METHODS: Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. RESULTS: Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. CONCLUSIONS: During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.

Diversity of nuclear protein fractions of hamster liver and hepatoma produced by DNaseI.

The structural and functional diversity between active and inactive chromatin is thought to depend, in part, upon differences in DNA-bound protein composition, including changes in the number of sulfhydryl groups. The aim of the present study was to compare protein composition in untreated nuclei, DNaseI-released and resistant nuclear fractions of hamster liver and Kirkman-Robbins hepatoma cells. Electrophoretic analysis of nuclear proteins showed some evident quantitative and qualitative differences between normal and neoplastic cells. The most significant diversities were noticed in DNaseI solubilized fraction of both types of cells. Nuclease attack released a characteristic set of non-histones with mol. wt 37,000, 50,000, 74,000 and 130,000-160,000 from transformed cells, and polypeptides of mol. wt 45,000 and 76,000 from normal cells. Cell-specific distribution within examined nuclear polypeptides was revealed using selective staining of their protein-bound sulfhydryls. Immunoblot analysis demonstrated that a non-histone protein with mol. wt 48,000, overexpressed in rodent tumour cells, was exclusively concentrated in liver DNaseI-sensitive fraction, which amounted only to 8.3% +/- 2.0% of total nuclear DNA. In hepatoma cells, however, this particular polypeptide is distributed between nuclease-sensitive and nuclease-resistant nuclear fractions. Non-histone protein of mol. wt 48,000 appeared to contain free sulfhydryl groups. In summary, these results show molecular specificity of nuclear proteins from normal and tumour cells and differences in their distribution among nuclease-released and nuclease-resistant nuclear fractions. The diversity in molecular characteristics and sulfhydryl group patterns observed among the examined proteins of normal and neoplastic cells may suggest their involvement in some changes in the rearrangement of nuclei during neoplastic transformation.

Characterization of proteins from chronic lymphocytic leukaemic cells: electrophoretic and immunological approach.

The protein composition of cellular fractions (nuclear, mitochondrial, microsomal and cytosolic) from normal and B-chronic lymphocytic leukaemia (CLL) cells isolated by differential centrifugation was analysed by SDS-polyacrylamide gel electrophoresis. Some diversities in electrophoretic characteristics of proteins from various compartments of normal and leukaemic cells were observed, mainly among nuclear proteins. Comparison of nuclear proteins from CLL cells of patients with different stages of the disease revealed that an expression of some specific for leukaemic cells components, especially a polypeptide (MW 46 kD) might be correlated with a stage of CLL. Immunoblotting analysis in the presence of antiserum raised against cancer-associated antigen, described as p65 indicated the association of this particular antigen with a nuclear fraction of leukaemic cells.

Nuclear proteins of hamster hepatoma after administration of antitumour agents.

One- and two-dimensional gel electrophoretic analysis of nuclear proteins of Kirkman-Robbins hepatoma was used to study the effects of the tumour growth inhibitors methotrexate (MTX) and acyclonucleoside (DHPQtB) on protein composition. MTX and DHPQtB inhibited Kirkman-Robbins hepatoma growth by 89.2 +/- 3.5% and 16.3 +/- 6.1% respectively. The biosynthesis and/or metabolism of some polypeptide spots was affected by these antitumour agents, especially among components with molecular wt/isoelectric points of 52,000-64,000/4.9-5.5, 69,000-78,000/5.0-5.9 and 88,000-100,000/5.1-5.9.

Hepatoma-associated nuclear matrix nonhistone antigens.

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.

Studies on low molecular weight nuclear protein of tumour and normal cells.

1. Preliminary results of comparative electrophoretical and immunological analyses of the components of two classes of non-histone proteins, i.e. NHP1 and NHP2 eluted from hydroxyapatite allowed to suppose that protein of Mw 18,000 is specific for animal tumour cells. 2. However, the studies on cellular distribution of this polypeptide indicated that it is exclusively located in nuclei of hepatoma and normal liver as well. 3. The former observation seems to be the result of changes of the affinity of this protein to DNA during neoplastic transformation.

The appearance of hepatoma-associated chromosomal non-histone proteins in rat liver after a single dose of 3'-MDAB followed by treatment of phenobarbital.

Previously we have described polyclonal antibodies that recognized a group of nuclear nonhistone proteins whose molecular weights ranged in size from 170 to 220 kDa. These antigenic nonhistone chromosomal proteins are abundant in rat hepatoma chromatin. In this report we discuss the synthesis and cellular localization of these particular proteins during the multistage process of hepatocarcinogenesis. The appearance of these antigenic proteins in rat liver nuclei approximately parallels the appearance of alpha-fetoprotein in the cytosol of hepatocytes. However, the immunoreactivity of antigenic proteins increased steadily even during the prominent dip in the AFP concentration between 50 and 100 days of carcinogenesis. The effect of the tumor promoting agent, phenobarbital, on the synthesis of antigenic nuclear proteins was also studied. The appearance of hepatoma-associated non-histone chromosomal proteins at early stages of tumor promotion during hepatocarcinogenesis was observed. The results of these studies demonstrate that the hepatoma-associated non-histone proteins are expressed not only in hepatoma cells, but also in hepatocyte cells committed to carcinogenesis.

Identification of a nuclear antigen with molecular weight of 48,000 differentially expressed in tumour and normal cells.

A non-histone protein with mol. wt of 48,000 differentially expressed in normal and tumour cells was identified using immunological criteria. Antibodies were raised against a component specific for Kirkman-Robbins hepatoma of mol. wt about 48,000 separated from hepatoma non-histone proteins by preparative electrophoresis in polyacrylamide gel. It was demonstrated by immunoblotting that Morris hepatoma 7777 and Ehrlich ascites cells share an antigenic non-histone protein with Kirman-Robbins hepatoma. Tumour cells when compared with normal cells, i.e. hamster and rat liver, are characterized by significant enrichment of this component. Intracellular distribution of the polypeptide with mol. wt 48,000 suggests that this component may be a structural protein the biosynthesis of which increases or the antigenic determinants of which change in tumour cells.

Hepatoma-associated non-histone proteins are phosphoproteins preferentially localized in nuclear matrix.

1. High molecular weight non-histone proteins (NHP) were isolated from Morris hepatoma 7777 by Sephadex G-100, S-200 chromatography. 2. Specific polyclonal antibodies were raised against these NHP in rabbits. These antibodies recognized specific NHP components present in Morris hepatoma 7777 and 8994, but not in normal rat liver. Hepatoma-associated antigens are phosphoproteins. 3. Immunologically specific NHP of Morris hepatoma are intensively concentrated in nuclear matrix fraction.

Molecular characterization of non-histone chromatin proteins from experimental tumours.

Using two-dimensional (2-D) electrophoresis, two non-histone chromatin protein fractions (NHCP1 and NHCP2) from three animal tumours (Kirkman-Robbins hepatoma, Morris hepatoma 7777 and Ehrlich ascites cells) and normal hamster liver were analyzed. Apart from many common components several tissue specific polypeptides of the NHCP1 and NHCP2 fractions were detected. It was found that some spots present in electropherograms of non-histone proteins of tumour cells (M X 10(-3)/pI): 17-24/4.9-6.5 (NHCP1 and NHCP2); 34-41/4.9-6.0 (HCP1 and NHCP2); 44-46/5.3-7.5 (HCP2); 46-49/5.0-7.5 (NHCP1); 49/5.9-7.5 (NHCP2) and 102-134/5.6-7.0 (NHCP1) were absent from normal liver.

Hamster liver chromatin immunospecific non-histone proteins.

Three antisera were prepared against non-histone protein classes named NHCP1, NHCP2 and dehistonized chromatin (with different affinity to DNA) from hamster liver. Two main antigenic bands of MW 17,000 and 36,000 were specific in the NHCP1 fraction and one antigen of MW 56,000 was specific for the NHCP2 fraction from nuclease-sensitive and especially nuclease-resistant chromatin. Other NHCP2 liver antigens of MW 22,000, 27,000, 30,000, 36,000, 37,000, 40,000, 45,000, 46,000, 51,000, 98,000 and 100,000 were present only in nuclease-resistant chromatin of hamster liver. Immunologically specific hamster liver non-histone proteins within the NHCP1 and NHCP2 fractions seem to be restricted to nuclease-resistant chromatin fraction of this tissue. The above mentioned liver specific antigens are absent or present only at trace amounts in analogous Kirkman-Robbins hepatoma fractions.