Multiplexed drug testing of tumor slices using a microfluidic platform.

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter.

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.

Stabilization and reactivation of the p53 tumor suppressor protein in nontumorigenic revertants of HeLa cervical cancer cells.

We demonstrated previously that loss of in vitro transformation and in vivo tumorigenicity in two independent revertant clones of HeLa cells (designated HA and HF) resulted from dominant-acting genetic changes. Analysis of the p53 tumor suppressor gene revealed stabilization and at least partial restoration of wild-type p53 transactivation properties pathways in both revertants of HPV-induced cell transformation. The half-lives of the p53 protein and both of the HA and HF clones were increased approximately 4 fold compared with the parental HeLa cells (16, 17, and 4 min, respectively). The levels of E6 viral protein expression were similar in the three cell lines, whereas the levels of the ubiquitin ligase protein, E6 associated protein (E6-AP), were elevated in the revertants. Western blot analysis of immunoaffinity-purified p53 demonstrated that stabilization of p53 in the revertants was correlated with a reduction in the in vivo formation of complexes involving the E6 oncoprotein and p53. Stabilization of p53 function in the revertants did not result from mutations in either the p53 or E6-AP genes. Despite the observed stabilization and restoration of p53 transactivation function in the revertants, exposure of the revertants to DNA-damaging agents did not result in elevated levels of p21(waf-1) protein and failed to induce growth arrest in the G1 phase of the cell cycle. However, p53-independent induction of p21(waf-1) protein also failed to induce the G1 phase of the cell cycle. Thus, restoration of wild-type p53 transactivation activity in the HA and HF revertants is insufficient to induce G1 arrest and reversion from HPV-induced cell transformation in our model system.

[The geterogenity of 137Cs and 90Sr distribution and dose loading on critical tissues of main seedling root]. / Geterogennost' raspredeleniia 137Cs i 90Sr i obuslovlennye imi dozovye nagruzki na kriticheskie tkani glavnogo kornia prorostkov.

It is shown, that the roots of plants concentrate 137Cs and 90Sr from water solutions in different zones: 137Cs--mainly in a meristem zone, 90Sr--in a stretching zone. The similar character of radionuclide distribution was established regarding water and soil cultures. The real dose loading on critical tissues of main root have appeared to be much higher than it was expected from the assumption of uniform distribution of radionuclides in tissues.

Alterations in H-ras1 promoter conformation during N-nitroso-N-methylurea-induced mammary carcinogenesis and pregnancy.

We previously demonstrated that H-ras1 oncogene mutations detected in N-nitroso-N-methylurea (NMU)-induced mammary tumors arose as background mutations within rat mammary cells (RMCs) and that NMU promoted the outgrowth of these preexisting mutants. We have now detected a putative DNA structure in the H-ras1 promoter of RMCs in vivo that was absent in NMU-induced mammary tumor cells. Analysis of the promoter in RMCs as a function of time after exposure to carcinogens indicated that NMU, but not 7,12-dimethylbenz(a)anthracene, initiated the loss of this structure with a half-life of 7 days. Although loss of the structure was irreversible in cells that gave rise to tumors, it was restored in normal RMCs by 120 days after exposure and was present in normal RMCs of animals bearing tumors, even 1 year after NMU exposure. The structure was also abrogated in RMCs during pregnancy and restored after lactation was terminated, suggesting that reversible regulation of the structure by hormones contributed to normal RMC growth. Thus, NMU may promote abnormal RMC growth by mimicking the effects of hormones on DNA conformation. We hypothesize that the NMU-induced alterations in promoter conformation irreversibly deregulates H-ras1 expression in initiated cells, thereby increasing the phenotypic penetrance of the conditional H-ras1 mutations.

Expression of neurotrophin genes in human fibroblasts: differential regulation of the brain-derived neurotrophic factor gene.

Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. We previously reported that BDNF and NGF gene expression are differentially regulated in mouse L929 fibroblasts. Here we examine expression of these three neurotrophins in human fibroblasts. Northern blots detected BDNF and NT-3 mRNAs in fibroblasts derived from lung (WI-38), calvarium and foreskin. WI-38 cells and foreskin fibroblasts expressed 1.6 kb as well as 4 kb BDNF mRNAs whereas only the smaller BDNF mRNA was detected in calvarium fibroblasts. NGF mRNA was present in foreskin and calvarium but not lung fibroblasts. In WI-38 cells serum treatment increased levels of BDNF mRNA within 2 hr. Cycloheximide did not inhibit the increase. Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) transiently suppressed BDNF mRNA. Treatment with both serum and TPA first stimulated and then transiently suppressed BDNF mRNA. TPA and/or serum did not significantly affect BDNF mRNA in calvarium fibroblasts. These results show that human fibroblasts derived from different tissues express and regulate neurotrophin genes differentially.

[The use of an immunohistochemical method for detecting N7-methylguanine in human cells]. / Ispol'zovanie immunogistokhimicheskogo metoda dlia vyiavleniia N7-metilguanina v kletkakh cheloveka.

An immunohistochemical method, followed by silvering (Ag-treatment) of the final product of peroxidase reaction for N7-methylguanine detection, was first employed for human cells treated in vitro with N-methylnitrosourea. This method appeared to be highly specific and sensitive, compared to the routine immunohistochemical technique. Evidence has been provided on the application of Ag-treatment for detecting N7-methylguanine in the esophagus epithelium of patients with a high risk of cancer with this particular locality.

[Increasing the sensitivity of an immunohistochemical method for detecting carcinogen adducts with DNA]. / Povyshenie chuvstvitel'nosti immunogistokhimicheskogo metoda vyiavleniia adduktov kantserogenov s DNK.

Application of polyclonal antibodies against N7-methylguanine (N7-MG) for immunohistochemical detection of the DNA damage following exposure to methylnitrosourea in cultured cells (CHO and IAR-27) has been shown. The approaches were applied: a) an extraction of nuclei, and b) a silver intensification of the end product of peroxidase reaction. Both the approaches were effective in increasing the sensitivity of immunohistochemical detection of N7-MG, the latter technique being most sensitive. It is proposed that the silver intensification technique is very perspective to have a high levels of sensitivity and specificity, a sufficient contrast and preservation of the cell morphology. The efficiency of the new approach in application to the purposes of molecular epidemiology is discussed.

Immunohistochemical detection of DNA alkylation adducts in rat and hamster liver after treatment with dimethylnitrosamine.

Monoclonal and polyclonal antibodies specific for methylation adducts have been applied in an immunohistochemical study of DNA damage in rat and hamster liver following exposure to dimethylnitrosamine. The approach was validated, for frozen and paraffin-embedded sections, by comparison with biochemical data on adduct levels in whole tissues or specific cell populations in the same experimental systems. The potential application of this method to human exposure assessment is discussed.

[Modifying effects of changes connected with pregnancy on 1,2-dimethylhydrazine-induced carcinogenesis in rats]. / Modifitsiruiushchee vliianie izmenenii, sviazannykh s beremennost'iu, na kantserogenez, indutsirovannyi 1,2-dimetilgidrazinom, u krys.

The modifying effect of pregnancy and lactation on carcinogenesis induced by single injection of 1,2-dimethylhydrazine to female rats 30 days before mating has been studied. The total incidence of malignant tumours in rats with pseudopregnancy, single pregnancy, pregnancy and lactation, repeated pregnancies was lower in comparison with virgin animals (75, 44, 68, 59 and 93%, respectively). Colon adenocarcinoma incidence in rats with single pregnancy or repeated pregnancies was lower than that in virgin rats (42, 49 and 76%, respectively). Protective effect was observed mainly in descending colon. The mesenchymal kidney tumours were not developed at all in rats with single pregnancy. In virgin animals it was 31%. The inhibition of tumour incidence in the liver (cholangioma, cholangiocellular carcinoma) was observed in rats with single pregnancy or pregnancy and lactation in comparison with virgin control (3, 5 and 24%, respectively).

[The hormonal sensitivity of a transplantable adenocarcinoma of the large intestine]. / Gormonochuvstvitel'nost' perevivnoi adenokartsinomy tolstoi kishki.

It was found that repeated transplantation of ACATOL strain cells resulted in a loss of their sensitivity to growth-stimulating effect of pentagastrin. The effect of gastrin receptor antagonist proglumide, on ACATOL cells growth was inconstant. ACATOL tumor was sensitive to VIP and glucagon in vitro (as shown by adenylate cyclase activity assay), that makes the model promising for selecting potent hormone drugs against colorectal cancer.

[Modifying effects of pregnancy and lactation on the structure of N-methyl-N-nitrosourea-induced tumors of the mammary glands in rats]. / Modifitsiruiushchee vliianie beremenosti i laktatsii na strukturu opukholei molochnykh zhelez, indutsirovannykh N-metil-N-nitrozomochevinoi u krys.

The structure of breast tumors induced by a single i. v. injection of 50 mg/kg methylnitrosourea was compared in virgin rats and those who had been given the carcinogen 30 days prior to onset of gestation. After delivery, progeny were weaned from some animals to stop lactation. In the parous animal group, epithelial component of benign tumors was more pronounced than in virgins whereas malignant tumors showed higher level of cell differentiation.

[An analysis of blood serum proteins in chronic alcoholism patients by a cross immunoelectrophoresis method]. / Analiz belkov syvootki krovi bolnykh khronicheskim alkogolizmom metodom perekrestnogo immunoélektroforeza.

Prealbumin, albumin, alpha 1-antitrypsin, alpha 1-antichymotrypsin, ceruloplasmin, haptoglobin, transferrin, IgG, IgM and IgA were studied in blood serum of healthy donors and of patients with chronic alcoholism by means of cross immuno-electrophoresis and immunodiffusion. Only content of alpha 1-antitrypsin was distinctly altered in blood serum of the patients with alcoholism as compared with normal state, while individual variations in content of the proteins studied were considerably higher in blood serum of the patients. At the same time, distinct dissimilarity of the patterns studied was found between healthy donors and patients with chronic alcoholism when concentration ratios of some positively and negatively charged acute phase proteins were calculated (alpha 1-antitrypsin/albumin, haptoglobin/albumin, haptoglobin/transferrin).

[Effect of pregnancy and lactation on the appearance of the carcinogenic action of N-methyl-N-nitrosourea in rats]. / Vliianie beremennosti i laktatsii na proiavlenie kantserogennogo deistviia N-metil-N-nitrozomocheviny u krys.

Female rats were injected intravenously with a single dose of 50 mg/kg N-methyl-N-nitrosourea (MNU). Those of the study group were mated with intact males 30 days later. Some progeny were weaned immediately after birth, and lactation ceased; however, the other females were allowed to feed. There was no relationship between gestation and lactation, on the one hand, and total incidence of tumors of all localizations, on the other, the latter being the same in both the study group and controls. Tumor multiplicity was higher in nullipara and nonlactating females. Gestation and lactation stimulated the incidence of tumors in the kidneys and large bowel as well as polyps in the uterus; however, they were associated with a lower frequency of adrenal adenomas and tumors in the forestomach.