The ubiquitin-proteasome system and its crosstalk with mitochondria as therapeutic targets in medicine.

The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.

ACTN3 Genotype in Professional Sport Climbers.

Ginszt, M, Michalak-Wojnowska, M, Gawda, P, Wojcierowska-Litwin, M, Korszen-Pilecka, I, Kusztelak, M, Muda, R, Filip, AA, and Majcher, P. ACTN3 genotype in professional sport climbers. J Strength Cond Res 32(5): 1311-1315, 2018-The functional RR genotype of the alpha-actinin-3 (ACTN3) gene has been reported to be associated with elite sprint/power athlete status. Although large and rapidly increasing number of studies have investigated the associations between the ACTN3 genotypes and athletic performance in various sport disciplines, there is a lack of studies on the genetic predisposition in sport climbing, which was selected to be part of the next Summer Olympic Games in Tokyo 2020 with three subdisciplines ("lead climbing," "speed climbing," and "bouldering"). The aim of the study is to determine the frequency distribution of ACTN3 genotypes and alleles in professional lead climbers and boulderers. 100 professional sport climbers from Poland, Russia, and Austria were divided into 2 equal groups: professional boulderers and professional lead climbers were involved in the study. ACTN3 allele frequencies and genotypes were compared with 100 sedentary controls. Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism method. The percent distribution of RR genotype in the boulderers was significantly higher than in lead climbers and controls (62 vs. 26%; 33%, respectively; χ = 17.230, p = 0.0017). The frequencies of ACTN3 R allele in boulderers differed significantly from lead climbers and controls (77 vs. 51%; 58%, respectively; χ = 15.721, p = 0.0004). The proportion of the ACTN3 RR genotype is significantly higher in boulderers than in lead climbers and may be related to the specific type of predisposition to this subdiscipline.

Recruitment of APPL1 to ubiquitin-rich aggresomes in response to proteasomal impairment.

Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.

The Genetic Variants of NOTCH3 (6746T>C) and PSMA6 (-8C>G) as Possible Risk Factors of Psoriasis Development.

Advances in genotypic technologies enable identification of possible associations between genetic variants of certain genes and increased risk of developing plaque psoriasis or psoriatic arthritis. The aim of the study was to analyze the NOTCH3 (6746T>C) (rs1044009) and PSMA6 (-8C>G) (rs1048990) polymorphisms and their role in genetic susceptibility to psoriasis. The study included 158 psoriatic patients and 100 healthy controls. The frequencies of the NOTCH3 genotypes differed between the psoriatic patients and healthy controls (p = 0.050). No differences were found in the distribution of PSMA6 genotypes and alleles between the psoriatic patients and healthy controls. The studied psoriatic patients presented a higher frequency of the CC genotype of PSMA6 compared to the healthy controls (8.8% vs. 2%, respectively). Psoriatic arthritis was more frequent among patients with the CC genotype of PSMA6 (p = 0.059). CC homozygosity of NOTCH3 was more commonly observed in the studied psoriatic patients than in the healthy controls (OR = 4.76, p= 0.032). The obtained data suggest that genetic variants of NOTCH3 (6746T>C) and PSMA6 (-8C>G) genes may play significant roles in psoriatic patients. Further studies are necessary to unequivocally determine their role as genetic risk factors of psoriasis development.

Endosomal adaptor proteins APPL1 and APPL2 are novel activators of beta-catenin/TCF-mediated transcription.

Canonical Wnt signaling regulates many aspects of cellular physiology and tissue homeostasis during development and in adult organisms. In molecular terms, stimulation by Wnt ligands leads to the stabilization of beta-catenin, its translocation to the nucleus, and stimulation of TCF (T-cell factor)-dependent transcription of target genes. This process is controlled at various stages by a number of regulatory proteins, including transcriptional activators and repressors. Here we demonstrate that the endosomal proteins APPL1 and APPL2 are novel activators of beta-catenin/TCF-mediated transcription. APPL proteins are multifunctional adaptors and effectors of the small GTPase Rab5, which localize to a subpopulation of early endosomes but are also capable of nucleocytoplasmic shuttling. Overexpression of APPL1 or APPL2 protein stimulates the activity of beta-catenin/TCF-dependent reporter construct, whereas silencing of APPL1 reduces it. Both APPL proteins interact directly with Reptin, a transcriptional repressor binding to beta-catenin and HDAC1 (histone deacetylase 1), and this interaction was mapped to the pleckstrin homology domain of APPL1. Moreover, APPL proteins are present in an endogenous complex containing Reptin, beta-catenin, HDAC1, and HDAC2. Overexpression of either APPL protein relieves Reptin-dependent transcriptional repression and correlates with the reduced amounts of HDACs and beta-catenin associated with Reptin as well as with the lower levels of Reptin and HDAC1 on the promoters of beta-catenin target genes. We propose that APPL proteins exert their stimulatory effects on beta-catenin/TCF-dependent transcription by decreasing the activity of a Reptin-containing repressive complex.

Signaling from endosomes: location makes a difference.

In all transmembrane receptor systems the kinetics of receptor trafficking upon ligand stimulation is maintained in a balance between degradative and recycling pathways in order to keep homeostasis and to strictly control receptor-mediated signaling. Endocytosis is commonly considered as an efficient mechanism of uptake and transport of membrane-associated signaling molecules leading to attenuation of ligand-induced responses. Accumulating evidence, however, shows that signaling from internalized receptors not only continues in endosomal compartments, but that there are also distinct signaling events that require endocytosis. Endocytic organelles form a dynamic network of subcellular compartments, which actively control the timing, amplitude, and specificity of signaling. In this review we provide examples in which signal transduction either requires an active endocytic machinery, or directly originates from various types of endosomes. Based on recent discoveries, we emphasize the close interdependence between signaling and endocytosis, and the physiological relevance of endocytic transport in health and disease.

Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex.

Multifunctional adaptor protein APPL1 [adaptor protein containing PH (pleckstrin homology) domain, PTB (phosphotyrosine binding) domain and leucine zipper motif] belongs to a growing group of endocytic proteins which actively participate in various stages of signalling pathways. Owing to its interaction with the small GTPase Rab5, APPL1 localizes predominantly to a subpopulation of early endosomes but is also capable of nucleocytoplasmic shuttling. Among its various binding partners, APPL1 was reported to associate with the nuclear co-repressor complex NuRD (nucleosome remodelling and deacetylase), containing both nucleosome remodelling and HDAC (histone deacetylase) activities, but the biochemical basis or functional relevance of this interaction remained unknown. Here we characterized the binding between APPL1 and NuRD in more detail, identifying HDAC2 as the key NuRD subunit responsible for this association. APPL1 interacts with the NuRD complex containing enzymatically active HDAC2 but not HDAC1 as the only deacetylase. However, the cellular levels of HDAC1 can regulate the extent of APPL1-NuRD interactions, which in turn modulates the nucleocytoplasmic distribution of APPL1. Increased binding of APPL1 to NuRD upon silencing of HDAC1 promotes the nuclear localization of APPL1, whereas HDAC1 overexpression exerts an opposite effect. Moreover, we also uncovered a NuRD-independent interaction of APPL1 with HDAC1. APPL1 overexpression affects the composition of the HDAC1-containing NuRD complex and the expression of HDAC1 target p21WAF1/CIP1. Cumulatively, these data reveal a surprising complexity of APPL1 interactions with HDACs, with functional consequences for the modulation of gene expression. In a broader sense, these results contribute to an emerging theme of endocytic proteins playing alternative roles in the cell nucleus.

The Association of GSTT1, GSTM1, and TNF-α Polymorphisms With the Risk and Outcome in Multiple Myeloma.

Oxidative stress and systemic inflammation are closely linked with increased risk of cancer development. Tumor necrosis factor alpha (TNF-α) is one of the pro-inflammatory cytokines. Glutathione S-transferases (GSTs) are enzymes involved in oxidative stress handling. Polymorphisms of genes encoding mentioned molecules may potentially influence the risk and the outcome in neoplastic diseases. Multiple myeloma (MM) is a hematological malignancy characterized by clonal, atypical plasma cell proliferation. In the present study we investigated the association of deletion polymorphisms in GSTT1/GSTM1 genes and single nucleotide polymorphisms (SNPs) in the TNF-α gene at positions -308/-238 with the risk and outcome in MM and sensitivity to bortezomib under in vitro conditions. One hundred newly diagnosed MM patients and 100 healthy blood donors were genotyped by means of multiplex PCR (for GSTs) and PCR-RFLP (for TNF-α). In a subgroup of 50 MM patients, bone marrow cells were treated with bortezomib in vitro. Patients with -238GA+AA or GSTT1-null genotypes had 2.0 (p = 0.002) or 2.29 (p = 0.013) fold increased risk of MM. The interaction effects and risk of MM were observed in GSTT1/GSTM1-null (OR = 2.82, p = 0.018), -308/-238GA+AA (OR = 5.63, p < 0.001), as well as in all combinations of -308 with GSTs. The -308/-238GA+AA genotypes in comparison to GG were associated with earlier MM onset-61.14 vs. 66.86 years (p = 0.009) and 61.72 vs. 66.52 years (p = 0.035), respectively. Patients with GSTM1-present had shorter progression-free-survival (15.17 vs. 26.81 months, p = 0.003) and overall-survival (22.79 vs. 34.81 months, p = 0.039) compared with GSTM1-null. We did not observe relationship between response rate and studied polymorphisms. The in vitro study revealed significantly higher number of apoptotic cells at 12 nM of bortezomib in GSTT1-present, GSTM1-null/present, -308GG and -238GG/GA+AA genotypes. Our findings comprise large analysis of studied polymorphisms in MM.

ACE Insertion/Deletion Polymorphism (rs4646994) Is Associated With the Increased Risk of Multiple Myeloma.

Introduction: The insertion (I allele) deletion (D allele) polymorphism of ACE gene (rs4646994) may influence the etiopathogenesis of multiple myeloma (MM). ACE gene is expressed in bone marrow cells and encodes angiotensin converting enzyme (ACE). It converts angiotensin I to active peptide angiotensin II, which stimulates proliferation of hematopoietic stem cells. This suggests possible association of ACE I/D gene polymorphism with MM. The aim of our study was to check possible impact of this polymorphism on risk of development and outcome of MM, as well as, sensitivity to bortezomib in cell cultures derived from MM patients. Objects and Methods: Genomic DNA from 98 newly diagnosed MM patients and 100 healthy blood donors were analyzed by PCR method. Chromosomal aberrations were detected by use of cIg-FISH. In a subgroup of 40 MM patients nucleated bone marrow cells were treated with bortezomib in vitro. Results: The Hardy-Weinberg equilibrium test showed that genotypic frequencies diverged significantly from the equilibrium. The differences between I and D allele frequencies in control and study population were significant (p = 0.046). We observed the association between DD genotype and more than 2-fold risk of MM - OR = 2.69; p < 0.0001. We did not detect any significant differences among studied genotypes regarding clinical and laboratory parameters. Moreover, we did not observe the association between survival of MM patients and I/D genotypes. Bortezomib increased number of apoptotic and necrotic cells, but the only statistically significant differences were observed in the number of viable cells at 1 nM between ID and DD genotypes (p = 0.026). Conclusion: Presented results confirmed the significant relationship between ACE (I/D) polymorphism and risk of MM development. We did not observe the association of ACE I/D polymorphism with disease outcome and bortezomib in vitro sensitivity.

Growth hormone signalling: sprouting links between pathways, human genetics and therapeutic options.

Our molecular understanding of growth hormone-induced signal transduction has improved significantly over the past decades. At the same time, human population genetics and the analysis of genetically engineered animals have led to the discovery of genes that control specific aspects of the overall growth process. Although, currently, growth disorders are still diagnosed and treated on empirical bases, it might soon be possible to stratify patients predominantly by genetic defect, with treatment based on our molecular understanding of the role of the affected gene in the disease.

Nuclear functions of endocytic proteins.

An increasing number of proteins appear to perform multiple, sometimes unrelated functions in the cell. Such moonlighting properties have been recently demonstrated for proteins involved in clathrin-mediated endocytosis. Some clathrin adaptors and endosomal proteins can undergo nucleocytoplasmic shuttling, which is often based on intrinsic sequence motifs and requires active transport mechanisms. Endocytic proteins can associate with nuclear molecules, changing their localization and/or activity and may modulate the levels and specificity of gene transcription. It is not clear how the nuclear and cytoplasmic pools of endocytic proteins are interconnected, or whether these molecules act as nuclear second messengers upon extracellular stimuli, but alike in endocytosis, they seem to form multi-component scaffolding platforms in the nucleus. Added to their endocytic functions, the nuclear roles of Eps15, Epsin1, CALM, HIP1, Dab1/2, beta-arrestins, APPL1/2 and the components of ESCRTs clearly increase the complexity of signaling networks affecting cellular growth, proliferation and homeostasis.

TP53 polymorphism in plasma cell myeloma.

INTRODUCTION: Significant and accessible predictive factors for bortezomib treatment in plasma cell myeloma (PCM) are still lacking. TP53 codon 72 polymorphism (P72R) results in proline (P) or arginine (R) at 72 amino acid position, which causes synthesis of proteins with distinct functions. The aims of our study were to: 1) analyze whether this polymorphism is associated with an increased risk of PCM; 2) study whether the P72R polymorphism affects overall survival (OS) among PCM patients; 3) assess the possible association of the P72R polymorphism with sensitivity to bortezomib in cell cultures derived from PCM patients. MATERIAL AND METHODS: Genomic DNA from newly diagnosed 59 patients (without IgVH gene rearrangements and TP53 deletions) and 50 healthy blood donors were analyzed by RFLP-PCR to identify TP53 polymorphism. Chromosomal aberrations were detected by use of cIg-FISH. The lymphocyte cell cultures from a subgroup of 40 PCM patients were treated with bortezomib (1, 2 and 4 nM). RESULTS: The P allele of the P72R polymorphism was more common than the R allele in PMC patients compared to controls (39% vs. 24%), and the difference was significant (p = 0.02). The PP and PR genotypes (in combina-tion) were more frequent among cases than in controls (65% vs. 42%, OR = 2.32, p = 0.04). At the cell culture level and 2 nM bortezomib concentration the PP genotype was associated with higher necrosis rates (10.5%) compared to the PR genotype (5.7%, p = 0.006) or the RR genotype (6.3%, p = 0.02); however, no effect of genotypes was observed at bortezomib concentrations of 1 and 4 nM. The shortest OS (12 months) was observed in patients with the PP genotype compared to patients with the PR or RR genotypes (20 months) (p = 0.04). CONCLUSIONS: The results suggest that P72R polymorphisms may be associated with an increased PCM risk and may affect OS of PCM patients. However, we saw no consistent results of the polymorphism effect on apoptosis and necrosis in cell cultures derived from PCM patients. Further studies are need in this regard.

Estimation of prognostic value of Bcl-xL gene expression in non-small cell lung cancer.

The aim of our study was to estimate the expression of the Bcl-xL gene, a member of Bcl-2 family, in NSCLC patients. A total of 60 consecutive patients diagnosed with NSCLC that underwent chemotherapy prior to surgery were reviewed. Bcl-xL expression was assessed on paraffin sections by in situ hybridization (ISH) and immunohistochemistry (IMH). We observed the presence of mRNA of Bcl-xL gene and its protein product overexpression in most patients (60 and 81.7%, respectively). In material examined no significant correlation was observed between the pattern of Bcl-xL or protein expression and any clinicopathological factors evaluated. The expression of Bcl-xL protein was low (less than 10% positive cells) in 11 patients (median survival time 29 months) as compared to 49 patients with overexpression (median survival time 21.0 months). The difference was not of statistic significance (p=0.27). In examined group the Bcl-xL mRNA was found in 36 patients, while it was absent in 24 cases. Median survival time was 14.5 and 86.5 months, respectively (p=0.001). In addition, 19.4% of 5-year survivals were achieved in patients with overexpression and 54.2% in patients with no mRNA present (p=0.002). The percentage of 5-year survival in patients with protein expression assessed by IMH was 30.6% (p=0.31). The estimation of Bcl-xL expression on mRNA and protein level was compared by the means of sign test and the significant difference was found (p=0.009). The inconsistency was related to 35% of cases. In comparison with IMH, ISH technique appeared to be more specific and accurate in assessment of 5-year survival (25 and 65%; 65 and 70%, respectively). The results of our study indicate that Bcl-xL mRNA overexpression may suggest poor prognosis in NSCLC.

ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors.

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin ß receptor (LTßR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTßR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

Endocytic proteins in the regulation of nuclear signaling, transcription and tumorigenesis.

Accumulating evidence argues that many proteins governing membrane sorting during endocytosis participate also in nuclear signaling and transcriptional regulation, mostly by modulating the activity of various nuclear factors. Some adaptors and accessory proteins acting in clathrin-mediated internalization, as well as endosomal sorting proteins can undergo nuclear translocation and affect gene expression directly, while for others the effects may be more indirect. Although it is often unclear to what extent the endocytic and nuclear functions are interrelated, several of such proteins are implicated in the regulation of cell proliferation and tumorigenesis, arguing that their dual-function nature may be of physiological importance.

Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth.

Several protein-tyrosine phosphatases (PTPs) have been implicated in the control of growth hormone receptor (GHR) signaling, but none have been shown to affect growth in vivo. We have applied a battery of molecular and cellular approaches to test a family-wide panel of PTPs for interference with GHR signaling. Among the subset of PTPs that showed activity in multiple readouts, we selected PTP-H1/PTPN3 for further in vivo studies and found that mice lacking the PTP-H1 catalytic domain show significantly enhanced growth over their wild type littermates. In addition, PTP-H1 mutant animals had enhanced plasma and liver mRNA expression of insulin-like growth factor 1, as well as increased bone density and mineral content. These observations point to a controlling role for PTP-H1 in modulating GHR signaling and systemic growth through insulin-like growth factor 1 secretion.

Expression of p21 and bcl-2 proteins in paraffin-embedded preparations of non-small cell lung cancer in stage IIIA after Etoposide and Cisplatin induced chemotherapy.

Apoptosis plays an important role in cancer pathogenesis. Several oncogenes and antioncogenes regulate this process. Loss of their normal function leading to cell resistance to apoptosis seems to be a key factor of neoplasm development. In tumour cells, programmed cell death is a spontaneous process and its intensity increases after chemo-, radio- and hormonotherapy. Amongst several genes and their products, bcl-2 and p21 genes play a significant role in the process. p21 gene product, cyclin-dependent kinase inhibitor, along with p53 gene take part in cell cycle regulation. Our study aimed at evaluating p21 and Bcl-2 protein expression in the cells of patients afflicted with stage IIIA of non-small cell lung cancer who underwent neoadjuvant chemotherapy (three courses of Vepesid and Cisplatin). Protein expression was evaluated in slides of tissue material obtained before pharmacological treatment (during bronchofiberoscopy) and after three courses of Vepesid and Cisplatin (during surgical tumour resection). Protein activity in tissue slides was conducted using histochemical method with labelled antibodies (immunoperoxidase staining procedure). The control material was obtained from patients who had not undergone inductive chemotherapy. The results were documented as photographs and presented as charts after extinction level measurement using cytophotometric technique. Decrease in Bcl-2 protein activity and increase in p21 protein level in tumour cells of patients after inductive chemotherapy were observed.

Expression of p53 gene in stage IIIA non-small cell lung cancer in patients after neoadjuvant chemotherapy with Vepesid and Cisplatin.

Apoptosis (programmed cell death) plays a very important role in the development regulation, homeostasis maintenance as well as in the origin of many diseases, including neoplasms. This process is genetically regulated and reflected in characteristic morphological and biochemical changes taking place in cells. The process is considered to be of great significance in tumour originating and growth as well as in tumour cell response to chemotherapy. There are many genes and their products that are involved in apoptosis. The following genes: p53, bcl-2 and p21 seem to have the greatest significance. Our study aimed at evaluating p53 gene expression in non-small-cell lung cancer patients after neoadjuvant chemotherapy. We examined the tissue material from 35 patients after three-cycle inductive chemotherapy (Vepesid and Cisplatin). The material was obtained before chemotherapy during bronchofiberoscopy and four weeks after drug treatment during surgery. The control group comprised patients who had not undergone inductive chemotherapy. After deparaffinising of tissue slides, gene p53 activity using in situ hybridisation technique was evaluated. Moreover, apoptosis valuation with TUNEL method was performed. The results were documented as photographs. Gene p53 activity level was estimated using cytophotometric technique. Our study revealed significantly higher percentage of cells undergoing apoptosis and increased gene p53 activity in tumour tissue slides of patients after neoadjuvant chemotherapy.

The preliminary estimation of bcl-2, bcl-X(L) and p53 genes expression in locally advanced non-small cell lung cancer.

Cell neoplastic transformation results from disturbances in expression of genes regulating its basic functions like cell cycle and apoptosis. Our paper presents preliminary estimation of expression of protooncogenes: bcl-2 and bcl-X(L) as well as of p53 suppressor gene in (NSCLC) non-small-cell lung cancer patients. The study comprised 16 NSCLC patients subjected to chemotherapy before operative procedure. Gene expression was evaluated on paraffin embedded specimens using in situ hybridization assay and immunohistochemical method. In the majority of examined patients, high expression of bcl-X(L) gene both at mRNA and protein level was ascertained. In the case of 10 patients (62.5%), higher p53 gene expression at mRNA level was observed, whereas higher level of P53 protein was determined only in four subjects (25%). In two of 16 cases (12.5%), protein product of bcl-2 gene was found, while in eight subjects (50%)--mRNA expression of the gene.