Cell Carcinoma Of Cervix Research Articles

Ubiquitin-Conjugating Enzyme UBE2Q2 Suppresses Cell Proliferation and Is Down-Regulated in Recurrent Head and Neck Cancer

The ubiquitin-proteasome system has a crucial role in maintaining and regulating cellular homeostasis including carcinogenesis. UBE2Q2, also designated Ubci, is one of the ubiquitin-conjugating enzymes (E2), and it has been reported that mRNA of UBE2Q2 is highly expressed in human head and neck squamous cell carcinoma, particularly hypopharyngeal carcinoma. However, the involvement of UBE2Q2 in carcinogenesis has not been fully elucidated. Most cases of head and neck carcinoma are treated with cis-diamminedichloroplatinum (II; CDDP) or docetaxel, which are the most effective chemotherapeutic agents against squamous cell carcinomas. Nevertheless, some head and neck cancers develop resistance to these drugs, although the causes and mechanisms remain unknown. In this study, we found high expression levels of UBE2Q2 in human head and neck carcinoma cell lines and cancer tissues by using an anti-UBE2Q2 antibody at the protein level. We also found that the expression level of UBE2Q2 is decreased in cell lines and cancer tissues that have resistance to CDDP or docetaxel and in cancer tissues treated with CDDP or docetaxel. Furthermore, we found that overexpression of UBE2Q2 affects cell proliferation and anchorage-independent cell growth. These findings suggest that UBE2Q2 is a novel oncosuppressor that inhibits tumor growth and is related to the resistance to anticarcinoma agents and that UBE2Q2 likely functions as a novel diagnostic tool and a potentially therapeutic target for head and neck squamous cell carcinoma.

Nanovesicles released by Dictyostelium cells: A potential carrier for drug delivery

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

Reprogramming of HeLa cells upon DNMT3L overexpression mimics carcinogenesis

Previously we had discovered loss of DNA methylation at the DNMT3L promoter, an enzymatically-inactive DNA methyltransferase, in squamous cell carcinoma of cervix indicating association between cancer and DNMT3L. This study extends this correlation further by identifying the role of DNMT3L in nuclear reprogramming, an event central to the process of carcinogenesis. We show that in cervical cancer cell lines, overexpression of DNMT3L, which functions by regulating the activity of DNMT3A and DNMT3B, increased cellular proliferation and anchorage-independent growth. Importantly, increased DNMT3L expression resulted in changed morphology of cells but this change was gradual and observed only after several passages. Interestingly, confluent cultures of DNMT3L-overexpressing HeLa cell colonies had characteristics of iPS cells. Concomitant with the morphological changes, expression pattern of genes important in nuclear reprogramming, development and cell cycle were observed to have significantly changed. Many imprinted genes, the known targets of DNMT3L, were downregulated. The slow nature of morphological changes and genome-wide nuclear reprogramming observed upon DNMT3L overexpression reinforces its role in carcinogenesis.

Antitumor activity of deoxypodophyllotoxin isolated from Anthriscus sylvestris: Induction of G2/M cell cycle arrest and caspase-dependent apoptosis

An active compound having antitumor activity was isolated from the root of Anthriscus sylvestris. Structural studies revealed that it was deoxypodophyllotoxin (DPPT), and its biological activity was evaluated in HeLa human cervix carcinoma cells. Flow cytometric analysis showed that DPPT arrests the cell cycle in the G2/M phase prior to apoptosis. The mechanisms of action of DPPT involve inhibition of tubulin polymerization, dysregulation of cyclin A and cyclin B1 expression, and activation of caspases-3 and -7.

Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant-field gel electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The dephosphorylation of DNA-PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 micromol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN-enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN.

Absence of STAT1 disturbs the anticancer effect induced by STAT3 inhibition in head and neck carcinoma cell lines

The family of signal transducers and activators of transcription (STAT) are transcription factors. Among them, STAT1 is associated with an apoptosis pathway, while STAT3 is associated with tumorigenicity in various cancer cells. In order to investigate the primary roles of STAT1 and STAT3 in head and neck squamous cell carcinoma (HNSCC), we blocked STAT3 with two JAK inhibitors: AG490 (JAK2-STAT3 pathway inhibitor) and JAK total inhibitor. When we inhibited STAT3 with AG490, significant cell death was observed. However, in the case of JAK kinase total inhibitor, no cell growth retardation was observed. We focused on the role of STAT1 in this phenomenon. Suppression of STAT1 by si-RNA resulted in increased cell survival. Furthermore, the growth inhibitory effect of AG490 was reduced by treatment with si-RNA of STAT1. These results reveal that STAT1 is required to promote the tumor killing effect of STAT3 inhibition in HNSCC.

Activation of p53 for the treatment of cancer

T umor suppressor p53 promotes cell death in response to environmental stress signals including DNA damage, hypoxia and aberrant proliferative signals like oncogene activation [Vazquez et al., 2008]. This molecule is important for combating carcinogenesis. A mutation inactivating p53 is found in about 50% of cancer including hepatoma, prostate cancer and lymphoma [Soussi and Wiman, 2007; Petitjean et al., 2007]. In cervical cancer cell line HelaS3, p53 activity is inhibited by human papillomavirus E6 and E7 proteins, which is demonstrated to be critical for the carcinogenesis of cervical cancer [Kessis et al., 1993]. Thus, activation of p53 is an appropriate approach for the treatment of cancers [Vazquez et al., 2008]. We read with great interest the paper ‘‘Roscovitine Up-Regulates p53 Protein and Induces Apoptosis in Human HeLaS3 Cervix Carcinoma Cells’’ published by WesieskaGadek et al. in your journal [Wesierska-Gadek et al., 2008]. Roscovitine increases p53 activity in HelaS3 cells through several mechanisms including increased protein expression, decreased degradation and protein activation of p53. The mechanism for the role of activated p53 in cancer treatment involves in both cell cycle arrest and apoptosis. Activation of p53 can increase p21 expression, which in turn inhibits a broad range of CDKs and PCNA-dependent DNA polymerase activity [Warbrick et al., 1997] and thus reduce cell cycle progression. Activation of p53 also causes cell apoptosis through activation of pro-apoptosis pathway and inhibition of IAPs (the inhibitors of apoptosis). Thus, activation of p53 can be used solely for the treatment of cancer. Roscovitine was compared with cisplatin and proved to be superior because roscovitine has no cytotoxicity. This research provided a new approach to activate p53. The authors showed that roscovitine phosphorylated p53 at Ser46 different from cisplatin which phosphorylated p53 at Ser15. The potency of roscovitine activating p53 is several folds lower than cisplatin. However, cisplatin at high dose causes cytotoxicity, neurotoxicity and nephrotoxicity because it makes DNA mutation. Roscovitine was

Growth inhibition of head and neck carcinomas by D‐allose

An inhibitory effect of D-allose, a rare sugar, on several cancer cell lines has been reported. This study aimed to investigate the growth inhibition of head and neck squamous cell carcinoma cells by D-allose. We treated 3 head and neck carcinoma cell lines with D-allose, D-fructose, D-psicose, and D-glucose. Cell growth assays as well as analyses of messenger RNA (mRNA) expression, cell cycle, apoptosis, and uptake of 14C-glucose were performed. D-allose had inhibitory effects on all 3 cell lines and tended to upregulate mRNA expression of glucose transporters, p21 and p53, and downregulate mRNA expression of cyclin A2, cyclin B1, and CDC2. We observed that D-allose tended to interfere with the intracellular uptake of D-glucose and induced apoptosis. Our results indicate that D-allose inhibits the growth of head and neck squamous cell carcinoma cells. D-allose has a considerable potential as a new anticancer agent in those patients.

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

BackgroundEnhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).Methodology/Principal FindingsMass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.Conclusions/SignificanceOur data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.

Expression of p16INK4A, CyclinD1, Ki-67 and HPV infection in cervical squamous cell carcinoma.

Objective To analyse the expression of p16INK4A, CyclinD1, Ki-67 proteins and HPV16/18, 31/33 in invasive squamous cell carcinoma of cervix(ISCC)and to indicate the significance of them in the occurrence and development of ISCC. Methods Using tissue microarray,in Situ Hybridization (ISH) and immunohistochemical method detected the expression of HPVI6/18, 31/33 and investigated the expression of p16INK4A, CyclinD1, Ki-67 in ISCC, CIN and NCE. Results was analysed by SPSS vision 13.0. Results The positive expression rate of HPV16/18, CyelinD1, Ki-67 in ISCC was markedly higher than that in CIN and NCE. Significant relationship was observed between p16INK4A protein expression and histological grade of the cancer. No significant correlation was observed between CyclinD1 protein expression and every clinical pathological parameters of ISCC. Conclusion High-risk HPV takes an important role in the development in ISCC. p16INK4A gene might not act as a suppressor-gene only on the occurrence in ISCC. Ki-67 can be looked as a valuable clinic index of diagnosing benign and malignant tumor. Key words: Uterine cervical neoplasms; Papillornaviriolae; Cyclin-dependent kinase inhibitor p16; CyclinD1; Ki-67 antigen

Smart delivery of antitumoral platinum complexes from biomimetic hydroxyapatite nanocrystals

This study widens the role of biomimetic hydroxyapatite (HA) nanocrystals as bone substitutes and describes how they can be used as bone-specific drug delivery devices for in situ treatment of bone tumors upon local implantation. The adsorption and release kinetics of bis-{ethylenediamineplatinum(II)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and bis-{ethylenediamineplatinum(II)}medronate on two kinds of HA nanocrystals having different morphologies, crystallinity degrees and surface areas have been investigated. The different chemical structures of the two Pt complexes appreciably affect not only the affinity towards the two kinds of HA, but also their release. The Pt complex loading is slightly greater for the HA characterized by lower crystallinity and higher surface area, with respect to the more crystalline one. The cytotoxicity of Pt complexes released from the HA were tested against human cervix carcinoma cells and, interestingly, were found to be more cytotoxic than the unmodified complexes. The released Pt species are therefore the active dichloridoethylenediamineplatinum(II) or related solvato species formed by Pt-bisphosphonate bond breaking.

Direct metallation of thienopyrimidines using a mixed lithium–cadmium base and antitumor activity of functionalized derivatives

A series of thieno[2,3-d]- and thieno[3,2-d]pyrimidines have been easily synthesized using as key step a deproto-cadmiation-trapping sequence. Some of the compounds thus synthesized were screened for anticancer (cytotoxic) activities, and (S)-2-(6-iodo-2-phenylthieno[2,3-d]pyrimidin-4-ylamino)-3-phenylpropanoic acid proved to have a significant activity towards liver, human breast and cervix carcinoma cell lines.

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.

Stabilization of gold(I) and gold(III) complexes by pyridil bis{3-hexamethylene-iminylthiosemicarbazone}: spectroscopic, structural and computational study

The reaction of pyridil bis{3-hexamethylene-iminylthiosemicarbazone} (H2Plhexim) with dichloro[2-(dimethylaminomethyl)phenyl-C1,N]-gold(III) ([Au(damp-C1,N)Cl2]) led to the isolation of the complexes [Au2(H2Plhexim)(Hdamp-C1)2Cl4]Cl(2).2H2O () and [Au(H2Plhexim)]2Cl2.S(8).6H2O (). The spectroscopic data for suggests that the bisthiosemicarbazone is coordinated to two different Au(III) atoms through the sulfur atoms. The fourfold coordination of the gold atoms is completed by one protonated Hdamp ligand, with the corresponding cleavage of an Au-N bond, and by two chloro ligands. The coordination mode and structure has been rationalized by means of DFT calculations. The crystal structure of shows the neutral bisthiosemicarbazone coordinated through the sulfur atoms in a linear S-Au-S geometry. Gold-gold interactions are not observed and this is due to the steric hindrance of the bulky groups present in the ligand, which prevent the approach of two neighboring gold atoms. During the reaction, the reduction of Au(III) to Au(I) takes place with the bisthiosemicarbazone oxidized to the elemental sulfur, S8, which is present in the complex. (H2O)12 clusters are also located in the void spaces. The in vitro antitumor activity against the HeLa-299 cell line was also investigated. The IC50 values are slightly higher than that of cisplatin. In comparison to the value for cisplatin, the maximum antiproliferative activity is higher in the Au(I) complex and similar in Au(III) complex, thus, the Au(I) complex exhibits greater activity against the human cervix carcinoma cell line.

A Simple Quartz Crystal Microbalance Nucleic Acid Sensor for Detection of Telomerase

A simple, fast, inexpensive sensitive/selective nucleic acid sensor based on the quartz crystal microbalance system (QCM) was developed for the detection of telomerase. A specially designed oligonucleotide probe, that is complementary to the template region of the telomerase enzyme, was immobilized onto the silver electrodes of piezoelectric crystals with polyethyleneimine adhesion cross-linked with glutaraldehyde. Optimum coating and immobilization conditions were determined. The QCM nucleic acid sensor was used for the detection of telomerase enzyme in human cervix carcinoma cell (HeLa) lysates. A significant difference between the telomerase negative cells (BHK) from HeLa cells, which was used for qualitative and quantitative detection of telomerase. An almost a linear correlation with the protein concentration was obtained with a R 2 value of 0.9621. The linear correlation coefficients (R2) for HeLa and BHK cell lysate obtained by applying Langmuir adsorption model, were 0.9630 and 0.9766, respectively. Telomerase activity was confirmed by stretch-PCR and fluorometric (F)-TRAP within HeLa extracts. The results obtained both with the QCM system developed in this study and F-TRAP method applied is significantly correlated (r = 0.968, p<0.001).

DNA binding property, nuclease activity and cytotoxicity of Zn(II) complexes of terpyridine derivatives

Two zinc(II) terpyridine complexes Zn(atpy)(2)(PF(6))(2) (1) (atpy = 4'-p-N9'-adeninylmethylphenyl-2,2':6,2''-terpyridine) and Zn(ttpy)(2)(PF(6))(2) (2) (ttpy = 4'-p-tolyl-2,2':6,2''-terpyridine) have been synthesized and characterized by elemental analysis, (1)H NMR and electrospray mass spectroscopy. The structure of complex 2 was also determined by X-ray crystallography, which revealed a ZnN(6) coordination in an octahedral geometry with two terpyridine acting as equatorial ligands. The circular dichroism data showed that complex 1 exhibited an ICD signal at around 300 nm and induced more evident disturbances on DNA base stacking than complex 2, reflecting the impact of the adenine moiety on DNA binding modes. Complex 1 exhibited higher cleavage activity to supercoiled pUC 19 DNA than complex 2 under aerobic conditions, suggesting a promotional effect of adenine moiety in DNA nuclease ability. Interestingly, both complexes demonstrated potent in vitro cytotoxicity against a series human tumor cell lines such as human cervix carcinoma cell line (HeLa), human liver carcinoma cell line (HepG2), human galactophore carcinoma cell line (MCF-7) and human prostate carcinoma cell line (pc-3). The cytotoxicity is averagely 10 times more active than the anticancer drug cisplatin.

Paclitaxel-loaded PEGylated PLGA-based nanoparticles: In vitro and in vivo evaluation

The purpose of this study was to develop Cremophor ® EL-free nanoparticles loaded with Paclitaxel (PTX), intended to be intravenously administered, able to improve the therapeutic index of the drug and devoid of the adverse effects of Cremophor ® EL. PTX-loaded PEGylated PLGA-based were prepared by simple emulsion and nanoprecipitation. The incorporation efficiency of PTX was higher with the nanoprecipitation technique. The release behavior of PTX exhibited a biphasic pattern characterized by an initial burst release followed by a slower and continuous release. The in vitro anti-tumoral activity was assessed using the Human Cervix Carcinoma cells (HeLa) by the MTT test and was compared to the commercial formulation Taxol ® and to Cremophor ® EL. When exposed to 25 µg/ml of PTX, the cell viability was lower for PTX-loaded nanoparticles than for Taxol ® (IC 50 5.5 vs 15.5 µg/ml). Flow cytometry studies showed that the cellular uptake of PTX-loaded nanoparticles was concentration and time dependent. Exposure of HeLa cells to Taxol ® and PTX-loaded nanoparticles induced the same percentage of apoptotic cells. PTX-loaded nanoparticles showed greater tumor growth inhibition effect in vivo on TLT tumor, compared with Taxol ®. Therefore, PTX-loaded nanoparticles may be considered as an effective anticancer drug delivery system for cancer chemotherapy.

Roscovitine up‐regulates p53 protein and induces apoptosis in human HeLaS3 cervix carcinoma cells

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.

Synthesis and in vitro antiproliferative activity of novel 1-benzhydrylpiperazine derivatives against human cancer cell lines

In order to explore the antiproliferative effect associated with the piperazine framework, several 1-benzhydrylpiperazine derivatives 8( a– d), 9( a– d) and 10( a– h) were synthesized. Variation in the functional group at N-terminal of the piperazine led to three sets of compounds, bearing the sulfonyl, amide and thiourea, respectively. Their chemical structures were confirmed by 1H NMR, LCMS, IR and elemental analysis. The antiproliferative effect of the compounds were evaluated in vitro using the MTT colorimetric method against one normal cell line (NF-103 skin fibroblast cells) and four human cancer cell lines (MCF-7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, four compounds exhibited interesting growth inhibitory effects against all four cell lines.

Nevirapine induces growth arrest and premature senescence in human cervical carcinoma cells

ObjectiveThe aim of this study was to assess the impact of nevirapine on a cervix carcinoma cell line. MethodsHeLa cells were cultured in Dulbecco's modified Eagle medium supplemented with 20% fetal bovine serum at 37 °C and humidified 10% CO2 in air. Nevirapine was purified from commercially available Viramune (Boehringer-Ingelheim), diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) in 350 µΜ final concentration and added to cell cultures 5 h after seeding. The same DMSO volume (0.2% final concentration) was added to controls. ResultsWe found that nevirapine treatment induces reversible growth arrest and produces morphological changes in treated cells. In contrast with previous reports the observed effects of nevirapine did not correlate with promotion of differentiation, but with induction of premature senescence. Premature senescence as a response to anti-tumour treatment is a common effect of the most anti-cancer chemotherapeutics. Nevirapine treated cells strongly accumulated SA-b-Gal activity and also expressed increased levels of p53 and p21 when analyzed via RT-PCR. In order to further explore the potent mechanisms of premature senescence induction we performed pChk2-Thr68 immunofluorescence analysis and found that nevirapine treated cells exhibited increased number of nuclear foci, indicating activated DNA damage response. ConclusionWe propose that at least in HeLa cell line nevirapine treatment exerts an effect far from the differentiation process, by introducing the cells into premature senescence. Based on these data, the effects of RT inhibitors should be further investigated since they may represent an additional agent against human cancer.