[Two Approaches to Cancer Development]. / Dva pohledy na vývoj nádoru.

BACKGROUND: The somatic mutation theory explaining the process of carcinogenesis is generally accepted. The theory postulates that carcinogenesis begins in a first renegade cell that undergoes gradual transformation from a healthy to a fully malignant state through the accumulation of genetic and epigenetic "hits". This theory focuses specifically on mutations and genetic aberrations, and their impact on cells. It considers tumors as populations of sick cells that lose control of their own proliferation. The theory was put forward by Robert Weinberg and Douglas Hanahan, and is the predominant view in current cancer biology. By contrast, the tissue organization field theory proposed by Carlos Sonnenschein and Ana Soto considers loss of physiological structure and function by a tissue as key events in tumor development. According to this theory, tumors arise at a tissue rather than at a cellular level. It is based on a presumption that proliferation status, rather than quiescence, is the default position of cells in multicellular organisms. AIM: The article aims to provide answers to following questions: Are the views of proponents of the somatic mutation theory (the reductionists) and proponents of the tissue organization field theory (the organicists) incompatible and incommensurable, even when the mainstream of tumor biology has shifted its attention from tumor cells toward the tumor microenvironment? Where to find a third interconnecting systemic approach? Is it useful to be aware of the controversy between reductionists and organicists? What this awareness contributes to? How do these alternative views influence practical oncology and tumor biology in general? CONCLUSION: Whether the true position is held by reductionists or organicists is unimportant. What is important is to be aware of the existence of these two concepts because this knowledge makes the way we think about tumor origin and development, and how we set up and interpret our experiments, more precise. KEY WORDS: carcinogenesis - mutation - cell - tissues - cell proliferation - cell quiescenceThis study was supported by grant of Internal Grant Agency of the Czech ministry of Health No. NT/13784-4/2012.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 29. 7. 2015Accepted: 27. 4. 2016.

[Brazilian story of the R337H p53 mutation]. / Brazilský príbeh mutace p53 R337H.

The p53 tumor suppressor is an evergreen of molecular oncology. Since its discovery in 1979, it has been subjected to intensive investigation. The p53 protein is composed of "only" 393 amino acid residues, and function of almost each of them has been addressed in detail. Somatic mutations are extremely frequent, they can be found almost in each of the p53 codons and in all types of tumors. Inherited p53 mutations are rare but very penetrant, and they are typically associated with development of a broad spectrum of tumors. However, in 2001, the p53 research provided an unexpected discovery: the R337H allele was found in southern Brazil. This allele was atypically associated with only one type of tumor -  childhood adrenocortical carcinoma and it exhibited low penetrance. Therefore, new data on functioning and impact of the R337H mutation were highly desired. The results obtained during a few following years helped to elucidate not only this specific p53 variant but also provided insight into general principles of mutant p53 variants function. It also turned out that all R337H alleles that are very frequent in southern Brazil originate from one common ancestor.

[Doublehit lymphomas -  review of the literature and case report]. / Lymfomy se dvema zásahy -  prehled literatury a kazuistika.

UNLABELLED: Blymphocytes are cells of the immune system responsible for the antibody  mediated immune response. As estimated, a human body can produce as much as 1011 specific antibodies. There are no specific genes coding for every individual antibody in the human genome. Discrepancy between the huge diversity of antibodies and limited coding capacity of the genome is solved by combination of unique arrangement of genetic information for immunoglobulin and unique genetic and somatic processes providing this wide spectrum of antibodies. On one side, these mechanisms represent a life protecting source of a wide spectrum of antibodies but at the same time, they can be life threatening by raising the risk of a serious tumor disease, the B cell lymphoma. Double hit lymphomas represent a specific group of B cell lymphomas often featuring concurrent rearrangements of BCL2 and MYC genes. Activation of the MYC oncogene, typical for Burkitt lymphoma (BL), causes strong stimulation of cell proliferation. High activity of BCL 2, typical for follicular lymphoma, induces resistance to apoptosis. Concurrent damage of regulation of apoptosis and proliferation is probably responsible for the typical clinical manifestation of double hit lymphomas - aggressive course, resistance to conventional chemotherapy, high-risk of early relapse, short overall survival, frequent extranodal and central nervous system involvement. Recently, these lymphomas have attracted a strong attention of researchers as they provide sharp insights into processes of lymphocytes maturing and lymphomas development and highlight the double edged nature of mechanisms allowing the antibody broad diversity. CASE REPORT: Fifty  three year  old man was diagnosed with B cell lymphoma unclassifiable with features intermediate between diffuse large B cell lymphoma (DLBCL) and BL, based on morphology and immunophenotype. Fluorescent in situ hybridization analysis revealed double hit lymphoma diagnosis as the tumor cells bear t(14;18) translocation concurrently with the MYC gene rearrangement. The patient died five months after dia-gnosis.

Roscovitine-induced apoptosis of H1299 cells depends on functional status of p53.

Roscovitine, an inhibitor of cyclin-dependent kinases, is promising anticancer agent. Its antiproliferative and cytotoxic effects can be mediated by the p53 signaling pathway. To define the role of p53 in roscovitine-induced cell response, we prepared H1299/p53 cell lines inducibly expressing specific variants of p53 (p53wt and hotspot R175H, temperature-dependent P98A, A159V, S215G, Y220C, Y234C mutants). In the presence of roscovitine, each cell line variant behaved in specific way reflecting activity of the p53 protein. Roscovitine decreased production of the cell cycle inhibitor p21 and induced apoptosis. This effect was the most efficient in cells expressing p53wt protein with full activity. The cell expressing partially and conditionally active p53 mutants responded to roscovitine less efficiently. The cells expressing p53 mutants A159V and Y234C were very sensitive to roscovitine but their response was clearly temperature-dependent. The cells expressing P98A, S215G and Y220C p53 mutants exhibited only weak sensitivity to roscovitine and underwent apoptosis in low frequency. In principle, each td p53 mutant responded to roscovitine in distinct way. We showed clearly that the impact of roscovitine on H1299 cells depends on functional status of p53 they produce. This suggests that patients with tumors exhibiting specific p53 variants can benefit from the roscovitine therapy.

[Lymphoma of the small intestine]. / Lymfomy tenkého streva.

A series of eight small intestine lymphomas comprised two cases of follicular lymphoma (FL), one anaplastic large cell lymphoma (ALCL) ALK negative, and five cases of diffuse large B-cell lymphoma. The lymphomas were diagnosed by routine hematoxylin-eosin staining, immunohistochemistry and the FISH method for translocation t(14;18). Immunohistochemistry revealed that the diffuse large B-cell lymphomas were of the non-germinal center type (non GC-DLBCL). In most cases, the tumors formed solid well-circumscribed nodules or resulted in diffuse infiltration of the intestinal wall. In one case of follicular lymphoma, microscopic foci of tumor were found in the intestinal mucosa which spread far from the primary nodule and probably beyond the resection border. It is difficult to ascertain whether this phenomenon represents colonization of pre-existing non-neoplastic follicles by lymphoma or spreading of the tumor within the same tissue. In this case, surgical removal of the lymphoma is problematic.

Identification of somatic hypermutations in the TP53 gene in B-cell chronic lymphocytic leukemia.

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P=0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase (AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

[Our experience in using fluorescence in situ hybridization FISH-Uro Vysion in diagnostics of urothelial carcinoma]. / Nase zkusenosti s metodou fluorescencní in situ hybridizace pri detekci uroteliálního karcinomu.

UNLABELLED: Urothelial carcinoma is a disease at high risk of recurrence after the initial therapy (70-80%) and with the tendency to progression accomplishing the recurrence (30%). Long lasting monitoring of patients with urothelial carcinoma is necessary. Cystoscopy and cytology are currently the primary modalities used to detect and monitor urothelial carcinoma. However, cytology has relatively poor sensitivity especially in well differentiated tumors. Cystoscopy is an invasive and relatively expensive method. Therefore, methods improving detection of urothelial carcinoma from urine specimens are employed. Uro Vysion (Vysis) fluorescence in situ hybridization (FISH) for improved detection of urothelial carcinoma was evaluated. MATERIALS AND METHODS: Bladder tumor progression is accompanied by increased chromosomal instability and aneuploidy of chromosomes 3, 7, 17 and loss of locus 9p21. A total of 124 patients were analyzed at Dpts. of Urology and Pathology, Faculty Hospital in Brno. Cytologically analyzed urine specimens were tested by FISH and simultaneously cystoscopy was employed including biopsy for histological examination. RESULTS: FISH analysis was positive in 35 cases, including 5 cases with negative biopsy and cytology. Negative FISH result was detected in 24 cases where the malignant status was determined. The sensitivity of FISH in our series was 58.9% and the specificity 88.1%. CONCLUSIONS: FISH is a relatively simple, speedy and non invasive diagnostic method. It detects the symptoms of malignity on the molecular level, which leads to earlier diagnosis and therapy and, hence, to potential extended survival. FISH makes it possible to take decision in cases of atypical or unclear cytological finding. The FISH method using the Uro Vysion kit appears as a prospective non invasive method capable of early UK detection, with a higher sensitivity than the standard cytology of urine.

Malignant fibrous histiocytoma of the parotid gland.

We described a rare malignant fibrous histiocytoma of the parotid gland (MFH) in a 63-year-old woman. During six months the tumour size became 10 cm in diameter with skin ulceration. The tumour was examined morphologically, by immunohistochemistry and molecular biology methods - FASAY and CGH. The histology revealed a storiform-pleomorphic type of MFH with high mitotic rate. The FASAY method identified a non-mutated p53 gene. The chromosomal changes were identified by the CGH method and 6 cytogenetic changes were found in the tumour cells (deletions at 8p12-p22, 13q32-qter, 14q24-qter, and gains of chromosomal material at 5p, 8q12-q23, and Xq25-qter). The patient died shortly after the beginning of chemotherapy. Autopsy revealed brain and cerebellar haemorrhage. No other tumour foci were proved. In view of short course of disease we lack the data about the influence of the non-mutated p53 gene on the prognosis and therapy.

[Waldenström macroglobulinemia--clinical manifestations and differential diagnosis and prognosis of the disease]. / Waldenströmova makroglobulinemie: klinické projevy a diferentiální diagnostika a prognóza nemoci.

Waldenström macroglobulinemia is defined by the presence of IgM type monoclonal immunoglobulin and histological prove of lymphoplasmocytary lymphoma in the bone marrow. Clinical symptoms of the disease depend on the pressure on the bone marrow by the malignant clone with subsequent cytopenia, extramedullary infiltration and toxic manifestations of monoclonal immunoglobulin. 6q deletion and absence of translocation in the sphere of the heavy immunoglobin chain gene, which is otherwise typical for other lymphoproliferative disorders, is the typical cytogenetic abnormality. The text describes the symptoms of the disease and the issues of its differential diagnosis.

The role of the carboxy terminus of v-Rel in transformation and activation of endogenous gene expression.

Proteins within the Rel/NF-kappa B transcription factor family can be divided into two functional domains, a homologous amino terminal region, the Rel Homology Domain, and a divergent carboxy terminal domain. The amino terminal sequences specify DNA binding, nuclear localization, and interaction with the I kappa B family of inhibitory proteins. The carboxy terminus of each protein functions as a transcriptional activation domain, however, precise definition of sequence requirements has been difficult. To further define these sequences, small 100 bp deletions were constructed throughout the carboxy terminus of v-Rel. Each resulting mutant was assayed for DNA binding, localization, protein complex formation, activation of endogenous gene expression and ability to transform bone marrow cells and fibroblasts. Surprisingly, deletion within the carboxy terminus had marginal effects on transforming potential. However, three separate regions were required for full activation of gene expression. Taken together, these results suggest that the carboxy terminus of v-Rel contains multiple sequences that participate in the activation of gene expression.

Retinoid X receptor suppresses transformation by the v-myb oncogene.

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.

Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

FASAY: a simple functional assay in yeast for identification of p53 mutation in tumors.

Alteration of the p53 tumor suppressor gene is the most common genetic defect known to occur in human tumors. Germ-line p53 mutations significantly increase the risk of developing diverse malignancies. FASAY is a simple functional assay for germ-line and somatic mutations in the p53 gene altering the transactivation capability of the p53 protein. The method was successfully used for mutation analysis of p53 in various cell lines, somatic tumor cells and blood cells. In addition, FASAY was also found effective as a tool for basic research of binding of mutant p53 proteins to promoters of different p53 target genes.

p53 status in breast carcinomas revealed by FASAY correlates well with p53 protein accumulation determined by immunohistochemistry.

The prognostic and predictive value of p53 mutation in breast cancer is still conflicting. The choice of the p53 status detection method may account for some discrepancies. In this pilot study we compared two differently-based methods for detection of p53 alteration in 32 breast carcinoma samples: the immunohistochemical method using Bp53, DO1 and DO11 monoclonal antibodies for analysis of the p53 protein accumulation in cell nuclei and the functional method FASAY. FASAY - functional analysis of the separated alleles in yeast - tests the capability of the human p53 to transactivate a reporter with a p53 binding site RGC driving the ADE2 gene in yeast. In our group the percentage of breast cancers with accumulated p53 protein was 50%, as well as percentage of mutant p53 scored by FASAY was 50%. Although the agreement of both methods, when comparing the results of individual patients was high (94%), our results show that immunohistochemistry does not reflect the p53 status quite exactly.

Gamma irradiation results in phosphorylation of p53 at serine-392 in human T-lymphocyte leukaemia cell line MOLT-4.

Exposure of human leukaemia MOLT-4 cells to ionizing irradiation led to apoptosis, which was detected by flow cytometric analysis and degradation of the nuclear lamina. The multiple signalling pathways triggered by either membrane or DNA damage play a critical role in radiation-induced apoptosis. The response to DNA damage is typically associated with the p53 protein accumulation. In this study, we proved that the transcriptionally active p53 variant occurs in the MOLT-4 cells and its abundance alteration is triggered in the gamma-irradiated cell population concomitantly with phosphorylation at both the serine-392 and serine-15 residues. The p21 upregulation followed the p53 phosphorylation process in irradiated MOLT-4 cells.

Effect of granaticin on the activity of DNA-directed RNA polymerase from Streptomyces granaticolor.

DNA-directed RNA polymerase was isolated from wild-type and mutant (asporogenic and granaticin overproducing) Streptomyces granaticolor. The effect of granaticin on the enzyme activity was compared with that of standard transcription inhibitors, rifampicin and rifamycin SV and shown to be zero.

Effect of protein phosphorylation on the activity of RNA polymerase in streptomycetes.

RNA polymerase was isolated from Streptomyces granaticolor and protein kinase was partially purified from Streptomyces albus. When RNA polymerase was treated with protein kinase in vitro the activity of RNA polymerase was markedly enhanced. Furthermore, a protein of M = 65 kDa was isolated which, after being phosphorylated, stimulated RNA polymerase activity in vitro. Because neither the beta-subunits nor the alpha-subunits of RNA polymerase were phosphorylated it is assumed that phosphorylation of the 65 kDa protein may regulate the activity of RNA polymerase in streptomycetes.

Isolation of DNA-dependent RNA polymerase from Streptomyces granaticolor and its binding to phage phi 29 DNA.

Partially purified DNA-dependent RNA polymerase of Streptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage phi 29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases from Escherichia coli and Bacillus subtilis.

[Clinical sequelae of mutation of the CBP gene]. / Klinické dusledky mutací genu CBP.

Gene CBP codes for a transcriptional coactivator, which can interact with many transcriptional factors. It modifies the process of transcription stimulated by these factors by specific binding to RNA polymerase II holoenzyme or by histone acetylation. CBP gene mutation is the molecular cause of autosomal dominant genetic disease called Rubinstein-Taybi syndrome that is manifested by mental and growth retardations, by typical face malformations and broad thumbs and broad big toes. The CBP gene can be affected by the t(8;16)(p11;p13.3) translocation resulting in production of the MOZ/CBP chimeric protein and in induction of acute myeloblastic leukaemia. Therapy using topoisomerase II inhibitors can induce the t(11;16)(q23;13.3) translocation causing acute myeloid or lymphoid leukaemia or myelodysplasia through production of the MLL/CBP protein chimera.

[Effect of products of germ-line and somatic gene mutations on chromatin remodeling]. / Dusledky vrozených a somatických mutací genu, jejichz produkty se úcastní prestavby chromatinu.

Chromatin remodeling is engaged in basic cell functions as DNA replication, recombination, DNA repair and transcription of genes. Chromatin is remodeled by ATP-dependent chromatin remodeling complexes and protein complexes covalently modifying histones. Germ-line mutations of the genes coding for proteins which participate in chromatin remodeling cause severe developmental diseases and they increase a risk of cancer development. Somatic mutations and translocations of such genes are associated with cancer development of certain tumors. Chromatin remodeling mechanisms can thus be targeted by special strategy of cancer therapy.