Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors, the most common sub-types are embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma. Immunohistochemical analysis revealed c-Myb expression in both eRMS and aRMS. c-Myb has been reported to be often associated with malignant human cancers. We therefore investigated the c-Myb role in RMS using cellular models of RMS. Specific suppression of c-Myb by a lentiviral vector expressing doxycycline (Dox)-inducible c-Myb shRNA inhibited proliferation, colony formation, and migration of the eRMS cell line (RD), but not of the aRMS cell line (RH30). Upon c-Myb knockdown in eRMS cells, cells accumulated in G0/G1 phase, the invasive behaviour of cells was repressed, and elevated levels of myosin heavy chain, marker of muscle differentiation, was detected. Next, we used an RD-based xenograft model to investigate the role of c-Myb in eRMS tumorigenesis in vivo. We found that Dox administration did not result in efficient suppression of c-Myb in growing tumors. However, when c-Myb-deficient RD cells were implanted into SCID mice, we observed inefficient tumor grafting and attenuation of tumor growth during the initial stages of tumor expansion. The presented study suggests that c-Myb could be a therapeutic target in embryonal rhabdomyosarcoma assuming that its expression is ablated.
- MeSH
- embryonální rhabdomyosarkom genetika metabolismus patologie MeSH
- G0 fáze * MeSH
- G1 fáze * MeSH
- genový knockdown MeSH
- karcinogeneze genetika metabolismus patologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protoonkogenní proteiny c-myb genetika metabolismus MeSH
- regulace genové exprese u nádorů * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Resveratrol is a phytoalexin that has been shown to inhibit cell proliferation of several cancer cell lines. In some cases this inhibition was specific for the transformed cells when compared with normal cells of the same tissue. To test whether this was the case in rat hepatocytes, we exposed primary rat hepatocytes in culture and transformed rat hepatic cells to this compound and studied its effect on cell proliferation, measuring deoxy-bromouridine incorporation and total DNA. We also studied the effect of resveratrol on the cell cycle of normal and transformed rat hepatocytes. We observed that resveratrol inhibited proliferation in a dose-dependent manner in both cases, with no differential action in the transformed cells compared to the normal ones. This compound arrested the cell cycle in G0/G1 in primary hepatocytes, while it arrested the cell cycle in G2/M in transformed cells. Transformed hepatocytes showed accumulation of cells in the S phase of the cell cycle.
- MeSH
- antioxidancia farmakologie MeSH
- buněčné dělení účinky léků MeSH
- DNA biosyntéza účinky léků MeSH
- G0 fáze účinky léků MeSH
- G1 fáze účinky léků MeSH
- G2 fáze účinky léků MeSH
- hepatocyty fyziologie účinky léků MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- proliferace buněk účinky léků MeSH
- stilbeny farmakologie MeSH
- transformované buněčné linie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
Cisplatin and doxorubicin are widely used anticancer drugs that cause DNA damage, which activates the ATM-Chk2-p53 pathway in cancer cells. This activation leads to cell cycle block or apoptosis, depending on the nature of the DNA damage. In an attempt to enhance the effects of these agents, we inhibited ATM/ATR and Chk2, which are known upstream regulators of p53. The cancer cell lines A2780 and ARN8, bearing the wild-type p53 protein, were used to study changes in p53 activation and trans-activation. Our results suggest that the G(1)-checkpoint, normally activated by DNA damage, is functionally overcome by the action of kinase inhibitors that sensitize cells to apoptosis. Both inhibitors show these effects, albeit with variable intensity in different cell lines, which is promising for other studies and theoretically for use in clinical practice.
- MeSH
- apoptóza MeSH
- cisplatina farmakologie MeSH
- DNA vazebné proteiny antagonisté a inhibitory metabolismus MeSH
- down regulace MeSH
- doxorubicin farmakologie MeSH
- G0 fáze MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové supresorové proteiny antagonisté a inhibitory metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- poškození DNA MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory metabolismus MeSH
- proteiny buněčného cyklu antagonisté a inhibitory metabolismus MeSH
- protinádorové látky farmakologie MeSH
- průtoková cytometrie MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Increased proportions of naive B cell subset and B cells defined as CD27(neg)CD21(neg)CD38(neg) are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of k deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27(neg)CD21(neg)CD38(neg) B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21(pos) naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27(neg)CD21(neg)CD38(neg) B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.
- MeSH
- antigen CD24 * biosyntéza genetika MeSH
- běžná variabilní imunodeficience * imunologie metabolismus patologie MeSH
- buněčná diferenciace imunologie MeSH
- dítě MeSH
- dospělí MeSH
- fenotyp MeSH
- G0 fáze * imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- počet lymfocytů MeSH
- podskupiny B-lymfocytů imunologie klasifikace patologie MeSH
- proliferace buněk MeSH
- regulace genové exprese * imunologie MeSH
- senioři MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
The interaction between retinoids and transforming growth factor-beta1 (TGF-beta1) leading to regulation of proliferation, differentiation and apoptosis is not still fully understood. In this study, we demonstrated that a combination treatment with all-trans retinoic acid (ATRA) and TGF-beta1 led to the enhancement of ATRA-induced suppression of cell proliferation, which is accompanied by inhibition of ATRA-induced apoptosis in human leukemia HL-60 cells. This effect was preceded by the arrest of cells in G0/G1 cell cycle phase linked with pRb protein dephosphorylation, continuous accumulation of p21 and transiently increased level of p27, inhibitors of cyclin-dependent kinases. Inhibition of ATRA-induced apoptosis by TGF-beta1 was associated with an increased level of Mcl-1 protein, an anti-apoptotic member of Bcl-2 family, but not with inhibition of mitochondrial membrane depolarization. Levels of other Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bad, Bak, Bax) were unaffected by simultaneous ATRA and TGF-beta1 treatment, when compared to ATRA alone. Upregulation of c-FLIP(L) protein, an inhibitor of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), correspond with inhibition of ATRA-induced (autocrine TRAIL-mediated) caspase-8 activation and apoptosis. These results suggest that apoptosis inhibition associated with proliferation block could depend on modulation of the TRAIL apoptotic pathway and regulation of the Mcl-1 protein level. In summary, we demonstrate that the balance of processes leading to regulation of proliferation and differentiation of myeloid cells can modulate cell sensitivity to apoptosis-inducing stimuli.
- MeSH
- aktivace enzymů účinky záření MeSH
- antigeny CD11b fyziologie účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- FLIP (buněčný) MeSH
- fosforylace MeSH
- G0 fáze účinky léků MeSH
- G1 fáze účinky léků MeSH
- granulocyty fyziologie účinky léků MeSH
- HL-60 buňky MeSH
- inhibitor p21 cyklin-dependentní kinasy biosyntéza účinky léků MeSH
- intracelulární signální peptidy a proteiny farmakologie metabolismus MeSH
- kaspasa 3 MeSH
- kaspasa 8 MeSH
- kaspasy metabolismus účinky léků MeSH
- lidé MeSH
- membránové glykoproteiny farmakologie metabolismus MeSH
- mitochondriální membrány fyziologie účinky léků MeSH
- nádorové buňky kultivované MeSH
- nádorové proteiny metabolismus účinky léků MeSH
- proliferace buněk účinky léků MeSH
- protein TRAIL MeSH
- protein X asociovaný s bcl-2 metabolismus účinky léků MeSH
- proteiny regulující apoptózu farmakologie metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- retinoblastomový protein metabolismus účinky léků MeSH
- synergismus léků MeSH
- TNF-alfa farmakologie metabolismus MeSH
- transformující růstový faktor beta farmakologie MeSH
- transformující růstový faktor beta1 MeSH
- tretinoin antagonisté a inhibitory farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. p27(Kip1) (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase. The level of p27 protein expression, usually high in G0/G1 resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G1 arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report). However, in addition to p27, several other Cdk inhibitors known to alter G1 progression coexist in most mammalian cells. To investigate the specific contribution of p27 in the control of the mitogen-sensitive G0/G1 arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serum-stimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.
- MeSH
- antisense DNA genetika MeSH
- buněčné linie MeSH
- Cricetulus MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- cykliny genetika fyziologie MeSH
- DNA biosyntéza MeSH
- G0 fáze genetika fyziologie MeSH
- G1 fáze genetika fyziologie MeSH
- inhibitor p21 cyklin-dependentní kinasy MeSH
- inhibitor p27 cyklin-dependentní kinasy MeSH
- inhibitory enzymů metabolismus MeSH
- komplementární DNA genetika MeSH
- křečci praví MeSH
- myši MeSH
- nádorové supresorové proteiny * MeSH
- proteiny asociované s mikrotubuly antagonisté a inhibitory genetika fyziologie MeSH
- proteiny buněčného cyklu * MeSH
- S fáze genetika fyziologie MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
