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Considering the important side effects of conventional microtubule targeting agents, more and more research focuses on regulatory proteins for the development of mitosis-specific agents. Polo-like kinase 1 (Plk1), a master regulator of several cell cycle events, has arisen as an intriguing target in this research field. The observed overexpression of Plk1 in a broad range of human malignancies has given rise to the development of several potent and specific small molecule inhibitors targeting the kinase. In this review, we focus on volasertib (BI6727), the lead agent in category of Plk1 inhibitors at the moment. Numerous preclinical experiments have demonstrated that BI6727 is highly active across a variety of carcinoma cell lines, and the inhibitor has been reported to induce tumor regression in several xenograft models. Moreover, volasertib has shown clinical efficacy in multiple tumor types. As a result, Food and Drug Administration (FDA) has recently awarded volasertib the Breakthrough Therapy status after significant benefit was observed in acute myeloid leukemia (AML) patients treated with the Plk1 inhibitor. Here, we discuss both preclinical and clinical data available for volasertib administered as monotherapy or in combination with other anticancer therapies in a broad range of tumor types. [ABSTRACT FROM AUTHOR]

Background: The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.Methods: After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.Results: HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan-Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.Conclusions: HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients. [ABSTRACT FROM AUTHOR]

Vaginal self-sampling may be valuable as an alternative method of cervical cancer screening in areas of poor resources, to enrol women who, otherwise, would not participate in population-based cervical cancer screening and in epidemiological follow-up studies. We assessed the reliability of mailed vaginal samples by evaluating the quantity and quality of genomic DNA in the samples. Mailed swabs ( n = 201) were compared with freshly collected samples ( n = 200) for DNA concentration (45.1 versus 50.9 ng/µl, respectively) and purity (mean optical density [OD] 260/280 ratio 1.88 versus 1.78, respectively). A small, non-significant, decrease in DNA yield with longer transport time was noted. The DNA yield of mailed samples was significantly lower compared to fresh samples ( P < 0.002), but this lower yield had little effect on polymerase chain reaction (PCR) amplification. In conclusion, the large majority of mailed self-sampled vaginal swabs resulted in DNA of adequate purity and concentration for further research. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis.Methods: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment.Results: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment.Conclusion: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway. [ABSTRACT FROM AUTHOR]

Ecteinascidin 743 (ET-743) is a new marine-derived agent with promising activity against a number of solid tumours. In four human tumour cell lines, the interaction between ET-743 and radiation was investigated in relation to the effects of ET-743 on the cell cycle, in vitro. Cell survival was measured based on quantitative staining of cellular protein by sulforhodamine B. A 24 h treatment with ET-743 before radiation resulted in a moderate increase in radiosensitivity in three out of four cell lines. Dose enhancement factors > or =1.8 were observed for concentrations resulting in 52, 46 and 30% cell kill in ECV304, H292 and CAL-27, respectively, whereas in A549 no radiosensitisation was observed (no significant increase in radiosensitivity). According to the combination index analysis, synergism was observed only in ECV304 and CAL-27 cells. A 24 h incubation with ET-743 resulted in a concentration-dependent G2/M block, which might explain the moderate radiosensitising effects in ECV304 and H292. The lack of radiosensitisation in A549 might be due to the S phase delay preceding the G2/M block at the moment of radiation, which only occurred in this cell line. In conclusion, ET-743 has moderate cell line-dependent radiosensitising properties; however, only when cytotoxic concentrations of ET-743 are used. In one of the four cell lines tested, no radiosensitisation was observed. [ABSTRACT FROM AUTHOR]

Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple restimulation cycles of peripheral blood lymphocytes with antigen-loeded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expreesing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments. [ABSTRACT FROM AUTHOR]

Adjuvant treatment of patients with colorectal cancer is hampered by a lack of reliable prognostic factors in addition to the clinicopathological staging system. A poorly defined but considerable fraction of Astler-Coller stage B patients will experience tumour recurrence, and some of the stage C patients will probably survive for a prolonged time after surgery without adjuvant treatment. Assessing parameters related to tumour angiogenesis has provided valuable prognostic information in different tumour types. The formation of new microvessels is part of the malignant phenotype in the majority of tumours. Alterations in tumour-suppressor genes, such as thep53 gene, or oncogenes, such as theras gene, have been found to be responsible for changing the local balance of pro- and antiangiogenic factors in favour of the former. In this prospective study, intratumoral microvessel density (IMD) was assessed by immunostaining tissue sections for CD31 and counting individual microvessels in selected and highly vascular regions in specimens of 145 colorectal cancer patients. p53 protein overexpression was semiquantitatively determined after immunohistochemistry. In both uni- and multivariate analysis, high IMD was significantly associated with shorter survival in the patients undergoing surgery with curative intent (Astler-Coller stages A-C). p53 added prognostic power to IMD, both in Astler-Coller stage B and stage C patients. An association between IMD and mode of metastasis was also noted. High IMD was strongly associated with the incidence of haematogenous metastasis during follow-up, but not with the presence of lymphogenic metastasis observed at surgery. This study confirms the results of previous retrospective analyses of IMD and survival in colorectal cancer and warrants a clinical validation by randomizing stage B tumour patients with high IMD and p53 overexpression between adjuvant treatment or not. [ABSTRACT FROM AUTHOR]

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-γ (IFN-γ) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CD1a+, HLA-DR+, CD4+, CD40+, CD80+ but CD14- cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-γ selectively induced HLA-DR and CD86 but did not up-regulate CD1a expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-γ could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-γ to GM-CSF plus TNF-α during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-α mutants, we investigated the relative involvement of TNF-α receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-R1 triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-α and IL-4; second, that the effect of TNF-α in DC generation involves signalling via the TNF-R1 receptor; and third, that IFN-γ counteracts some of the effects of IL4 in DC generation. [ABSTRACT FROM AUTHOR]

The effects of both 13-cis-and all-trans retinoic acid (RA) on colony formation of normal bone marrow (BM) progenitors were investigated in semi-solid (methylcellulose) assays, using either isolated CD34+ cells or BM mononuclear cells. Single cell liquid cultures were performed to further discriminate between direct and indirect effects. RA action results in significant decrease of colony forming units (CFUs). This effect is more pronounced starting from CD34+ progenitors than starting from total BM. This overall decrease in CFUs is due to selective inhibition of CFU-M (macrophage) and erythroid colonies (BFU-E). At the single cell level the CFU-M inhibition is confirmed with--in addition--a significant inhibition of CFU-GM (granulocyte-macrophage) and a marked stimulation of CFU-G (granulocyte)s. Both retinoids exert the above-mentioned effects. All-trans RA, however, is effective at a tenfold lower concentration (10(-7)M) than 13-cis RA (10(-6)M). Results on CD34+ BM fractions (substantially reduced in accessory cells) demonstrate that the described effects can probably be attributed to the direct action of RA on these progenitors; single progenitor (CD34+) cell liquid cultures further prove this point. [ABSTRACT FROM AUTHOR]

The effects of both 13- cis-and all- trans retinoic acid (RA) on colony formation of normal bone marrow (BM) progenitors were investigated in semi-solid (methylcellulose) assays, using either isolated CD 34+ cells or BM mononuclear cells. Single cell liquid cultures were performed to further discriminate between direct and indirect effects. RA action results in significant decrease of colony forming units (CFUs). This effect is more pronounced starting from CD 34+ progenitors than starting from total BM. This overall decrease in CFUs is due to selective inhibition of CFU-M (macrophage) and erythroid colonies (BFU-E). At the single cell level the CFU-M inhibition is confirmed with - in addition - a significant inhibition of CFU-GM (granulocyte-macrophage) and a marked stimulation of CFU-G(granulocyte)s. Both retinoids exert the above-mentioned effects. All- trans RA, however, is effective at a tenfold lower concentration (10 M) than 13- cis RA (10 M). Results on CD 34+ BM fractions (substantially reduced in accessory cells) demonstrate that the described effects can probably be attributed to the direct action of RA on these progenitors; single progenitor (CD 34+) cell liquid cultures further prove this point. [ABSTRACT FROM AUTHOR]

Human dendritic cells (DC) are highly professional antigen presenting cells for the priming of naive cytotoxic T cells. Gene transfer in DC would be a useful strategy to load DC with relevant de novo synthesized antigens for immunotherapeutical purposes. As a first step towards a DC-based gene therapy, we examined the efficiency of nonviral transfection in different types of cultured human dendritic cells with a humanized red-shifted green fluorescent protein reporter gene. Plasmid DNA transfection by electroporation or lipofection was used to transfect CD34+ progenitor cell-derived DC (PC-DC) and Langerhans’ cells (PC-LC), as well as monocyte-derived DC (Mo-DC). While lipofection was unsuccessful in all types of DC, we obtained high-efficiency gene transfer by electroporation in PC-LC (16%) and PC-DC (12%). In contrast, electroporation was strikingly less efficient in Mo-DC (2%). The potent allostimulatory capacity of DC was still retained in electroporated PC-DC and PC-LC. In conclusion, electroporation of antigen expressing plasmid DNA is an efficient tool for nonviral gene transfer in PC-DC and PC-LC, but not in Mo-DC and could be useful for the development of DC-based tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Examines the association between Chlamydia trachomatis (CT) and human papilloma virus (HPV) infection in a study that controlled for sexual behavior. Use of SPSS package version 11.5 in performing stepwise logistic regression analysis; Examination of the history of sexually transmitted disease diagnoses, emergency contraception use and unintended pregnancy, marital status, education and parity; Prevalence of HPV in women with CT infection.

This article focuses on a study related to HPV in cervix and vagina. Cervical cancer screening by Papanicolaou smear has shown its use in reducing both incidence and mortality. Nowadays, cervical tumors are mostly diagnosed in women who were not, or not properly, screened. The invasive sampling method of screening is one of the reasons why women do not participate. The efficiency of cervical cancer screening could be increased if a less invasive test were available. This study aimed to investigate HPV prevalence in cervix and vagina on samples taken by a professional.

Background: Based on their different mechanisms of action, non-overlapping side effects and radiosensitising potential, combining the antimetabolites pemetrexed (multitargeted antifolate, MTA) and gemcitabine (2',2'-difluorodeoxycytidine, dFdC) with irradiation (RT) seems promising. This in vitro study, for the first time, presents the triple combination of MTA, dFdC and irradiation using various treatment schedules.Methods: The cytotoxicity, radiosensitising potential and cell cycle effect of MTA were investigated in A549 (NSCLC) and CAL-27 (SCCHN) cells. Using simultaneous or sequential exposure schedules, the cytotoxicity and radiosensitising effect of 24 h MTA combined with 1 h or 24 h dFdC were analysed.Results: Including a time interval between MTA exposure and irradiation seemed favourable to MTA immediately preceding or following radiotherapy. MTA induced a significant S phase accumulation that persisted for more than 8 h after drug removal. Among different MTA/dFdC combinations tested, the highest synergistic interaction was produced by 24 h MTA followed by 1 h dFdC. Combined with irradiation, this schedule showed a clear radiosensitising effect.Conclusions: Results from our in vitro model suggest that the sequence 24 h MTA --> 1 h dFdC --> RT is the most rational design and would, after confirmation in an in vivo setting, possibly provide the greatest benefit in the clinic. [ABSTRACT FROM AUTHOR]

Background: Vinca alkaloids are an important class of anticancer agents and semisynthetic vinca alkaloids are developed to improve the therapeutic index of this class of drugs. In the present study, a direct comparison was made between vinflunine and vinorelbine regarding their radiosensitizing and cell cycle effects.Methods: Four human tumour cell lines were tested under identical experimental conditions, using equitoxic concentrations of vinflunine and vinorelbine.Results: Vinflunine and vinorelbine induced a comparable radiosensitizing effect (p-value never below 0.01) when cells were incubated for 24 h immediately prior to radiation. Regarding the cell cycle effects, a statistically significant concentration-dependent G2/M block was seen after 24 h incubation with vinorelbine in all tested cell lines. Similar results, with small cell line-related differences, were observed with vinflunine.Conclusion: The radiosensitizing effects of both semisynthetic vinca alkaloids were comparable (not statistically different) and nearly always cell line-specific and concentration-dependent. The cell cycle effects could be related to the observed radiosensitizing effects. Considering the more favourable toxicity profile of vinflunine, this agent might be more promising than vinorelbine for chemoradiation studies in the clinic. [ABSTRACT FROM AUTHOR]