Characterization of three B-cell lymphoma cell lines from chemotherapy resistant patients with respect to in vitro sensitivity to 21 antitumor agents,ABC-transporter expression and cellular redox status

PURPOSE: The aim of this study was to characterize three new, recently established non-Hodgkin lymphoma cell lines (GUMBUS, DOGUM, and DOGKIT), isolated from patients developing high-clinical resistance to cytotoxic therapy, with respect to sensitivity toward 21 antitumor drugs from different classes of action, expression of three ABC transporters: P glycoprotein (Pgp) (MDR1 and ABCB1), multidrug resistance related proteins (MRP1) (ABCC1), and MRP2 (ABCC2), as well as a range of antioxidative enzymes and glutathione (GSH). The results were compared to analogous data from the well-known HL-60 and U-937 cells. METHODS: The MTT assay was used to measure cell growth inhibitory activity. Transporter expression was determined by using an electrophoresis/Western blot system. GSH and enzyme activities were measured by employing functional assays with photometric detection. Pre-incubation with hydrogen peroxide was chosen as a model for oxidative stress. RESULTS: Based on the 50% growth inhibitory values (GI(50) values) of 21 known antitumor agents, the cell lines were sensitive again to chemotherapeutics after being in culture for at least 15-18 weeks. DOGUM and DOGKIT were most sensitive toward antitumor drugs in in vitro cytotoxicity assays while DOGUM was the least sensitive. None of the cell lines expressed measurable levels of any of the three transporters investigated and showed only moderate variation in their antioxidative defense system. After pre-treatment with hydrogen peroxide, GSH peroxidase (GPx) activity increased and, in general, a decrease in the growth inhibitory activities of various platinum antitumor agents occurred. Furthermore, all three cell lines rapidly acquired resistance to doxorubicin, methotrexate, and cisplatin again in vitro after only 3-5 treatment cycles with the respective drug. CONCLUSIONS: The therapy-resistant lymphoma cell lines GUMBUS, DOGUM, and DOGKIT were sensitive to antitumor agents once again after they had been established in culture. However, their sensitivity to antitumor agents can be rapidly decreased in vitro by either introducing the cells to culture conditions favoring oxidative stress or by exposing the cells at regular intervals to an antitumor drug. The ability of these three cell lines to quickly adapt to toxic insults in their environment is probably the reason why clinical resistance occurred.     

Intestinal parasitoses and environmental factors in a rural population of Argentina, 2002-2003

We evaluate the prevalence of intestinal parasites in 504 people and the degree of association between environmental variables and parasites found in population, soil and water in a rural area of Argentina during 2002-2003. A structured survey was used to evaluate the environmental variables and fecal-human, soil and water samples were analyzed. The prevalence of parasites was 45.4%. Most prevalent protozoa were Blastocystis hominis (27.2%) and Giardia lamblia (6.9%), while the most prevalent helminth was Ascaris lumbricoides (3.8%). The analyzed environmental variables showing association (p < 0.05) with presence of parasites in population were: cardboard-tin or wooden house, dirt floor, home or communal water pump, faucet outside the house or public faucet and cesspool or latrine. Parasite forms were found in 82.3% of the soil samples and in 84.2% of the water samples. In both samples we found parasites that were also found in people. In this study we have found deficient sanitary conditions associated with presence of parasites in population and we have evidenced that contaminated soil and water were the source of these parasites.     

Is the quantity of circulatory cell-free DNA in human plasma and serum samples associated with gender,age and frequency of blood donations?

Circulatory cell-free DNA (cf-DNA) is increased in a variety of clinical pathologic conditions; therefore, these markers could be widely used as markers for detecting and monitoring several disorders. To better understand the biology of this molecule, we analysed the relationship between the level of circulatory cf-DNA and physiological parameters such as gender, age and frequency of blood donations. Paired plasma and serum samples were obtained from 87 blood donors and 50 healthy adults who had never donated blood. Cf-DNA was extracted from plasma and serum samples using the MagNA Pure LC Instrument. Quantity determination of circulatory cf-DNA was performed by TaqMan real-time PCR for the ubiquitous GAPDH gene. Our data showed that the concentration of cf-DNA in serum was about eightfold higher than that in plasma. Regarding the level of these circulatory species, no significant differences were observed between the age-matched men and women and gender-matched, different-age cohorts, except in women who were older than 60 years of age. Frequent blood donations did not increase the circulatory species. Circulatory cf-DNA in plasma and serum samples is not correlated with human gender and human age except in women who are older than 60 years of age. Frequent blood donation did not affect the quantity of circulatory cf-DNA. The explanation for the latter most likely is the short half-life time of free fetal DNA in maternal circulation.     

Preparation and biological evaluation of 111In-, 177Lu- and 90Y-labeled DOTA analogues conjugated to B72.3

Three 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) analogues were evaluated for relative in vivo stability when radiolabeled with (111)In, (90)Y and (177)Lu and conjugated to the monoclonal antibody B72.3. The DOTA analogues evaluated were "NHS-DOTA" [N-hydroxysuccinimdyl (NHS) group activating one carboxylate], "Arm-DOTA" (also known as MeO-DOTA; with a p-NCS, o-MeO-benzyl moiety on the methylene group of one acetic acid arm) and "Back-DOTA" (with a p-NCS-benzyl moiety on a backbone methylene group of the macrocycle). The B72.3 was conjugated to the DOTA analogues to increase the retention time of the radioloabeled conjugates in vivo in mice. The serum stability of the various radiometalated DOTA conjugates showed them to have good stability out to 168 h (all >95% except (111)In-NHS-DOTA-B72.3, which was 91% stable). Hydroxyapatite stability for the (111)In and (177)Lu DOTA-conjugates was >95% at 168 h, while the (90)Y DOTA-conjugates were somewhat less stable (between 90% and 95% at 168 h). The biodistribution studies of the radiometalated DOTA-conjugates showed that no significant differences were observed for the (111)In and (177)Lu analogues; however, the (90)Y analogues showed lower stabilities, as evidenced by their increased bone uptake relative to the other two [2-20% injected dose per gram (% ID/g) for (90)Y and 2-8% ID/g for (111)In and (177)Lu]. The lower stability of the (90)Y analogues could be due to the higher beta energy of (90)Y and/or to the larger ionic radius of Y(3+). Based on the bone uptake observed, the (177)Lu-NHS-DOTA-B72.3 had slightly lower stability than the (177)Lu-Arm-DOTA-B72.3 and (177)Lu-Back-DOTA-B72.3, but not significantly at all time points. For (90)Y, the analogue showing the lowest stability based on bone uptake was (90)Y-Arm-DOTA-B72.3, perhaps because of the metal's larger ionic radius and potential steric interactions minimizing effective complexation. The (111)In analogues all showed similar biological distributions at the various time points. This study suggests that care must be taken when evaluating (90)Y-labeled antibodies and in using NHS-DOTA-antibody conjugates with (177)Lu. All evaluations should be extended to time points relevant to the half-life of the radiometal and the therapy applications.     

Interphase fluorescence in situ hybridization analysis detects a much higher rate of thyroid tumors with clonal cytogenetic deviations of the main cytogenetic subgroups than conventional cytogenetics

In benign thyroid lesions, three main cytogenetic subgroups, characterized by trisomy 7 or structural aberrations involving either chromosomal region 19q13.4 or 2p21, can be distinguished by conventional cytogenetics (CC). As a rule, these aberrations seem to be mutually exclusive. Interphase fluorescence in situ hybridization (I-FISH) analysis on benign as well as malignant thyroid neoplasias has been performed in the past, but rarely in combination with CC. In the present paper, we have analyzed 161 benign thyroid lesions both with CC and I-FISH on touch preparations by using a multi-target, triple-color FISH assay as well as dual-color break-apart probes for detection of the main cytogenetic subgroups. Within the samples, I-FISH detected tumors belonging to either of the subgroups more frequently than CC (23 vs. 11.4%), either due to small subpopulations of aberrant cells or to cryptic chromosomal rearrangements (three cases). Thus, I-FISH seems to be more sensitive than CC, particularly in the detection of subpopulations of cells harboring cytogenetic aberrations that may be overlooked by CC. In summary, I-FISH on touch preparations of benign thyroid lesions seems to be a favorable method for cytogenetic subtyping of thyroid lesions.