The aim of the project proposed is to test the immunogenicity of genetic and peptide vaccines both individually and in different combinations as well as in combination with chemotherapy. The aim is also to propose a therapeutic vaccine suitable to be used in human patients. In parallel, a prospective clinical study will aim to determine the immune profile of CML patients and its changes in the course of the disease.

Cílem projektu je prověřit imunogennost několika typů genetických a peptidových vakcín , a to samostatně, v různých kombinacích a rovněž v kombinaci s chemoterapií, a posléze navrhnout terapeutickou vakcínu vhodnou pro humánní aplikaci. Cílem prospektivní klinické studie bude určit imunologický profil pacientů s CML a jeho proměny v průběhu nemoci.

• MeSH
• chronická myeloidní leukemie farmakoterapie   terapie   MeSH
• filadelfský chromozom MeSH
• imunoterapie metody   trendy   MeSH
• mortalita MeSH
• myši MeSH
• translokace genetická MeSH
• transplantace hematopoetických kmenových buněk MeSH
• tyrosinkinasy aplikace a dávkování   terapeutické užití   MeSH
• vakcíny MeSH
• Check Tag
• myši MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• hematologie a transfuzní lékařství
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

• MeSH
• bcr-abl fúzní proteiny genetika   imunologie   MeSH
• časové faktory MeSH
• chronická myeloidní leukemie genetika   imunologie   patologie   prevence a kontrola   MeSH
• DNA vakcíny genetika   imunologie   MeSH
• HL-60 buňky MeSH
• imunizace MeSH
• lidé MeSH
• mapování epitopu MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidové fragmenty imunologie   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

B210 cells are murine (BALB/c) cells transformed by bcr-abl fusion gene. After intravenous administration they are capable of inducing leukaemia-like disease in syngeneic mice. From these cells a thymidine-kinase less subline was derived. It was significantly less pathogenic than the parental cells. However, a highly pathogenic clone denoted B210cTK-/cl-2 was isolated from its population. As determined by Western blotting, these cells produced more p210 protein than the parental B210 cells. To successfully transfect these cells a modified electroporation method was introduced. Bicistronic plasmids carrying gene for herpes simplex thymidine kinase (HSV TK) and the gene for either granulocyte-monocyte colony stimulation factor (GM-CSF), interleukin-2 (IL-2) or interleukin 12 (IL-12) were used for the transfection experiments. Gradually, cell lines producing these cytokines were isolated in media supplemented with hypoxantin, aminopterin and thymidine (HAT). All of them were highly sensitive to ganciclovir in vitro confirming that the cells produced HSV TK. The genetic modification of B210cTK-/cl-2 was associated neither with the alteration of p210 bcr-abl production nor with any changes in expression of MHC class I molecules. From populations of each of the three lines several cell clones were isolated and tested for the production of the respective cytokines. The original uncloned population and several clones differing in the cytokine production were administered intravenously into mice. All animals survived without symptoms of the disease suggesting that the gene-modification was associated with the loss of pathogenicity. Keywords: CML, Bcr-Abl, HSV TK, cytokines, gene-modified tumour cells, pathogenicity.

• MeSH
• adjuvancia imunologická genetika   MeSH
• bcr-abl fúzní proteiny analýza   MeSH
• cytokiny biosyntéza   genetika   MeSH
• ganciklovir terapeutické užití   MeSH
• geny abl MeSH
• MHC antigeny I. třídy analýza   MeSH
• MHC antigeny II. třídy analýza   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• thymidinkináza genetika   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Two mouse HPV16-transformed cell lines, viz. MK16 cells, which induce metastasizing tumors, and TC-1 cells, which induce non-metastasizing tumors were transduced with the gene for mouse endostatin. Two clones constitutively expressing endostatin were isolated from each of them. They were denoted ME3 and ME9, and TE2 and TE5, respectively. When inoculated into mice, ME3 cells were non-oncogenic. Nearly all mice inoculated with ME9 cells developed tumors, but considerably later than did the parental MK16 cells and metastasis formation was strongly reduced in these animals. On the other hand, TE2 and TE5 cells displayed oncogenic potential similar to that of the parental cells. To provide more information on these different effects of endostatin production, cell lysates of all six lines studied were tested for the content of 25 factors known to be involved in angiogenesis. The parental MK16 cells differed from the parental TC-1 cells and also from all endostatin producing sublines by a markedly higher production of interleukin 1alpha (IL-1alpha) and, to a lesser extent, by a higher production of several other factors tested. Additional experiments indicated that the suppression of the production of IL-1alpha by the parental MK16 caused by endostatin was due to an autocrine mechanism.

• MeSH
• autokrinní signalizace MeSH
• časové faktory MeSH
• endostatiny genetika   metabolismus   MeSH
• endoteliální buňky metabolismus   MeSH
• geny ras MeSH
• interleukin-1alfa metabolismus   MeSH
• interleukin-2 genetika   metabolismus   MeSH
• kultivační média speciální metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádory plic genetika   imunologie   metabolismus   prevence a kontrola   sekundární   virologie   MeSH
• onkogenní proteiny virové genetika   MeSH
• Papillomavirus E7 - proteiny MeSH
• proliferace buněk MeSH
• represorové proteiny genetika   MeSH
• transdukce genetická MeSH
• transformované buněčné linie MeSH
• virová transformace buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

For our experiments we selected two oncogenic, bcr-abl-transformed mouse cell lines, viz. B210 and 12B1. Both cell types are capable of inducing leukemia-like disease in syngeneic BALB/c mice after intravenous inoculation. 12B1 cells can moreover form solid tumors after subcutaneous injection. Since immunotherapy would expectedly be most effective in animals in which the tumor mass had been reduced by other therapeutic means, we attempted to develop a combined therapeutic system for suppressing tumor growth. In the present study, mice inoculated with the aggressive 12B1 cells were treated with imatinib mesylate (IM), mouse interferon alpha (IFNalpha) and cyclophosphamide (Cy) in combination with genetically modified tumor cells engineered to produce various cytokines. These cell vaccines had been derived from B210 cells. Therapy with IM or IFNalpha alone or cell immunotherapy alone resulted in partial suppression of tumor growth. Of the different therapeutic regimens tested, a combination of repeated doses of IM, IFNalpha and cell vaccines with one relatively high dose of Cy (200 mg/kg) was the most effective, resulting in tumor-free survival of a large portion of mice. The spleens, livers and bone marrows of the successfully treated animals were tested for the presence of bcr-abl-positive cells by means of RT-PCR technique. Results were negative, this suggesting that the animals had been cleared of residual disease.

• MeSH
• bcr-abl fúzní proteiny imunologie   terapeutické užití   MeSH
• cyklofosfamid aplikace a dávkování   MeSH
• experimentální nádory imunologie   terapie   MeSH
• faktor stimulující granulocyto-makrofágové kolonie biosyntéza   MeSH
• financování organizované MeSH
• imunoterapie metody   MeSH
• interferon gama aplikace a dávkování   MeSH
• interleukin-12 biosyntéza   MeSH
• interleukin-2 biosyntéza   MeSH
• kombinovaná terapie MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• piperaziny aplikace a dávkování   MeSH
• polymerázová řetězová reakce MeSH
• protinádorové látky aplikace a dávkování   MeSH
• protinádorové vakcíny imunologie   MeSH
• pyrimidiny aplikace a dávkování   MeSH
• transformované buněčné linie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

One of the gene therapy strategies in oncology is immunization with cancer cells that express various cytokines. We used a thymidine-kinase deficient (cTK-) cell line designated 123IA, which had been derived from HPV16-transformed mouse (C57BL/6) cells MK16/I/III/ABC (MK16). To obtain genetically modified cells, 123IA cells were transfected with bicistronic plasmid vectors carrying the herpes simplex type 1 thymidine kinase (HSV TK) gene and either the gene for the mouse B7.1 (CD80) co-stimulatory molecule or the gene for the monocyte-chemoattractant protein 1 (MCP-1). For control purposes, a plasmid vector carrying only the HSV TK gene was used. The transfected cells were cultivated in medium supplemented with hypoxanthine, aminopterin and thymidine. For comparative purposes we also used B9 cells, which express the granulocyte-macrophage colony stimulation factor (GM-CSF) and had been derived from 123A cells by transduction with the recombinant adeno-associated virus carrying the HSV TK gene and the mouse GM-CSF gene. All of the cell lines isolated were found to be sensitive to minute amounts of ganciclovir, revealing the production of HSV TK, and to express the respective transgenes. When inoculated into 5-week-old female syngeneic mice, cells expressing either GM-CSF or B7.1 were non-oncogenic. On the other hand, nearly all mice inoculated with MCP-1-producing cells developed tumours, though considerably later than animals inoculated with the same dose of the parental MK16 cells. Animals injected with GM-CSF- or B7.1-producing cells were protected against challenge with the parental MK16 cells. When another mouse (C57BL/6) HPV16-transformed oncogenic cell line, TC-1, which differs from the MK16 cells in a number of properties such as MHC class I and B7.1 expression, was used for the challenge, the protective effect was much less pronounced.

• MeSH
• antigeny CD80 genetika   MeSH
• buněčné linie MeSH
• chemokin CCL2 genetika   MeSH
• faktor stimulující granulocyto-makrofágové kolonie genetika   MeSH
• financování organizované MeSH
• ganciklovir farmakologie   MeSH
• geny MHC třídy I MeSH
• imunizace MeSH
• lidský papilomavirus 16 genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• onkogenní proteiny virové genetika   MeSH
• protinádorové vakcíny imunologie   MeSH
• represorové proteiny genetika   MeSH
• transfekce MeSH
• transformované buněčné linie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

The role of stem cells in cancer formation and spreading has been established. As with normal tissue, the cancer stem cells need a special microenvironment to support their growth. This microenvironment may be represented by the tumor stroma. One of the possible ways of tumor stromal formation is the epithelial-mesenchymal transition of tumor epithelium. Following this mechanism, stromal cells must share the basic genetic alterations with the tumor cells. In an attempt to create a system capable of testing some aspects of the mesenchymal cell-keratinocyte interactions, we studied the effects of the fibroblastoid mouse TC-1 cells that were prepared by the introduction of human papillomavirus type 16 (HPV16) genes E6 and E7 to lung epithelial cells on the phenotype of normal human interfollicular and hair follicle keratinocytes. From this point of view, they may resemble stromal cells formed by the epithelial-mesenchymal transition of cells from HPV-induced squamous cell carcinoma. In contrast to 3T3 murine embryonic fibroblasts which were used as control cells, TC-1 cells influenced not only the size of the keratinocytes and the shape of their colonies, but also induced the expression of keratins 8 and 19 and vimentin. In conclusion, TC-1 cells exhibited a marked biological activity by influencing the behavior of the normal human follicular and intefollicular keratinocytes. This observation is compatible with the hypothesis that stromal cells play an important role in tumor progression and spreading.

• MeSH
• buňky 3T3 MeSH
• buňky stromatu fyziologie   MeSH
• fenotyp MeSH
• fibroblasty fyziologie   MeSH
• financování organizované MeSH
• keratin-8 genetika   MeSH
• keratinocyty cytologie   MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádory MeSH
• velikost buňky MeSH
• vimentin genetika   MeSH
• vlasový folikul cytologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

• MeSH
• genetická terapie MeSH
• nádory terapie   MeSH