Dual oligopeptides modification mediates arsenic trioxide containing nanoparticles to eliminate primitive chronic myeloid leukemia cells inside bone marrow niches.

Chronic myeloid leukemia (CML) is one type of hematopoietic stem cell diseases. Although BCR-ABL1 tyrosine kinase inhibitors are remarkably effective in inducing remission in chronic phase patients, they are not curative in a majority of patients due to their failure to eradicate residual CML stem/progenitor cells, which reside in bone marrow niches. Here, we presented novel dual oligopeptides-conjugated nanoparticles and demonstrated their effective delivery of arsenic trioxide in bone marrow niches for the elimination of primitive CML cells. We encapsulated As-Ni transitional metal compounds into polymeric nanoparticles based on the reverse micelle rationale. The loading density and stability of arsenic trioxide in nanoparticles were improved. In vitro experiments demonstrated that dual oligopeptides conjugated nanoparticles could deliver arsenic trioxide into bone marrow niches including endosteal niches and vascular niches. The colony-forming activity of CML cells was remarkably restrained in the presence of metaphyseal bone fragments pre-incubated with bone marrow niche targeted arsenic nanoparticles. The in vitro vascular niche model suggested that CML cell proliferation was also successfully inhibited through a tight contact with HUVECs, which were pre-treated using niche-targeted arsenic nanoparticles. This bone marrow niche targeted delivery strategy has a potential usage for the treatment of CML and other malignant hematologic disorders originated from the bone marrow.

Hidden stressors in the clonogenic assay used in radiobiology experiments.

While clonogenic assays are extensively used in radiobiology, there is no widely accepted procedure for choosing the composition of the cell culture media. Cell line suppliers recommend a specific culture medium for each cell line, however a researcher will frequently customize this aspect of the protocol by supplementing the recommended support medium with additives. For example, many researchers add antibiotics, in order to avoid contamination of cells and the consequent loss of data, with little discussion of the influence of the antibiotics on the clonogenic survival of the cells. It is assumed that the effect of any variables in the growth medium on cell survival is taken into consideration by comparing the survival fraction relative to that of controls grown under the same conditions. In the search for better cancer treatment, the effect of various stressors on clonogenic cell survival is under investigation. This study seeks to identify and test potential stressors commonly introduced into the cell culture medium, which may confound the response to radiation.

The sensitivity of the MDA-kb2 cell in vitro assay in detecting anti-androgenic chemicals--identification of sources of variability and estimation of statistical power.

In vitro assays for anti-androgens have been developed as screening tools for the identification of androgen receptor (AR) antagonists. We explored the usefulness of such assays for experimental purposes that require quantitation of effects in a highly reproducible manner, such as multi-component mixture experiments or evaluation of extracts of complex environmental samples. We have investigated sources of experimental variation in the MDA-kb2 assay for AR-antagonists. By omitting phenol red from culture media, avoiding media changes and extending the period allowed for cell attachment, the dynamic range increased. Variations in luminescence readings decreased, with smaller coefficients of variation within- and between-experiments. Normalisation of luminescence values to positive controls improved experiment-to-experiment reproducibility and allowed pooling of data from independent experiments. We also performed statistical power analyses to determine the minimal suppression of androgenic (DHT) effects by test agents that are detectable as statistically significantly different from positive controls (so-called minimum significant differences, MSD). Using the modified assay protocol extensive concentration-response analyses were conducted with bisphenol A, BDE100 and vinclozolin. Our modified procedure improves considerably the reproducibility of the MDA-kb2 assay.

Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model.

In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

In vitro anti-tumor activity of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone against six established human cancer cell lines.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and its influence on six human cancer cell lines. Among SMMC-7721, 8898, HeLa, SPC-A-1, 95-D and GBC-SD cell lines, SMMC-7721 cells was the most sensitive one in these tested cell lines, with IC50 equal to 32.3 +/- 1.13 microM, EC50 equal to 9.00 +/- 0.36 microM and the therapeutic index equal to 3.59. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 9 microM DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid SMMC-7721 cells were 49.44 +/- 1.06% after 48 h treatment with 18.0 microM DMC. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. To our knowledge, this is the first report on anti-tumor activity by DMC.

Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture.

The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation. Contaminations of autologous blood stem cells with cancer cells have been described. Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR. Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy. Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy. Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR. RNA was extracted by standard methods and reverse transcription was performed with MMV-RT. Integrity of RNA was checked by coamplification of housekeeping sequences. Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently. A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture. Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry. Ten leukaphereses from 6 women were available for PCR-analysis and cell culture. Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells. No sample was positive for cultured cells and negative for CK19-mRNA. Overall, the results corresponded in 80%. Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80%. PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.

Assessing sensitivity in suspension to cytosine arabinoside: statistical analysis, associations with clinical outcome and experimental design.

As part of the treatment protocol in 40 leukaemia patients using cytosine arabinoside for remission induction therapy, the sensitivity of each patient's blast cell progenitors to the drug was determined by the liquid suspension assay. To summarize the dose-response curves obtained in this assay, we considered a model that assumes the existence of a resistant subpopulation of progenitor cells. We also considered the median effect model popular in pharmacokinetics. Both models were similar in their ability to describe the data. Parameter values that characterize a low baseline number of progenitor cells exhibiting high sensitivity both at low and high dosages were found to indicate good prognosis. We also identified a simple, economical, robust and efficient design for conducting future assays.

The delay before onset of accelerated tumour cell repopulation during radiotherapy: a direct maximum-likelihood analysis of a collection of worldwide tumour-control data.

The worldwide collection of control data for head and neck tumours presented by Withers et al. (Withers, H.R., Taylor, J.M.G. and Maciejewski, B. Acta Oncol. 27: 131-146, 1988) was reanalysed using a model which includes an explicit lag phase before the onset of tumour clonogen repopulation. A direct maximum-likelihood approach was used and the methodology extended to include the computation of profile-likelihood confidence limits. A statistically significant (p = 0.02) lag of 29 days was obtained with 95% confidence limits covering the range 17-31 days. However, the confidence interval was disconnected, and excluded the period 21-23 days. The analysis gave a time factor of 0.66 Gy/day. The mean values confirm the conclusions drawn by the original authors using a two-stage (indirect) method, and the values are similar to those calculated here for another data set comprising 496 patients (lag period = 26 (19-33) days). However, the data set itself is retrospective, and potentially subject to a number of biases. Therefore any clinical conclusions can only be tentative. A new feature of the methodology is the computation of profile-likelihood confidence limits and this will be useful in future direct analyses of clinical data of this type. The more usually computed normal approximation to the confidence limits have been shown to be inadequate in this analysis, and either profile-likelihood limits or likelihood ratio tests must be employed to determine the significance of the model parameters.

A statistical model for in-vitro assessment of patient sensitivity to cytotoxic drugs.

Formation of colonies in semisolid medium is an assay used for the study of stem cell characteristics in hematopoietic and solid tumors. Previous experience with leukemia patients failed to show an association between the reduction in colony formation observed when patient blast cells were exposed to increased concentrations of an anticancer agent, and the subsequent patient response to the agent. By introducing a model that takes into account the possibility of a resistant subpopulation of clonogenic cells, the paper demonstrates that the null result was due to an inadequate summarization of the dose-response curve, and in fact a statistically and biologically significant association exists between one of the parameters of the model and patient response. The properties, implementation, and interpretation of the model are discussed.