Non-uniformity in in vitro drug-induced cytotoxicity as evidenced by differences in IC50 values - implications and way forward.

Cell lines have proven indispensable for in vitro experiments and their utility as experimental models range from understanding the fundamental cell functioning to drug discovery. One of the most common utility of cell lines is for in vitro drug testing. Drug testing involves determining the cytotoxic effects of the drugs and such a measurement is expressed as the IC50 values of drugs. Although determination of IC50 values of drugs on cell lines is one of the most common in vitro experimental approaches, a significant amount of variations can be observed in the results obtained from such studies. Although the variations in the IC50 values of a drug on different cells lines can and should vary, the non-uniformity of such results reported from different studies using a particular drug on a specific cell line is a matter of concern. We present the IC50 values of 5 most commonly used drugs 5-fluorouracil, bleomycin, cisplatin, doxorubicin and methotrexate obtained from several in vitro cell line-based studies. Some of the factors which contribute to the non-uniformity of the IC50 values for a particular drug from different studies are discussed as three types of factors, the biological, non-biological and human factors. Also, ways in which such variations can be reduced to obtain universally common, reliable results are presented.

Biochemical Mediators and Mechanisms Involved in Chromatin Conformational Remodeling through the Cell Cycle.

Chromatin undergoes structural remodeling through the cell-cycle stages. Remodeling of the chromatin structure is extremely important for events occurring during these stages. The five major levels of structural organization, from the double-strand structure to the metaphase chromosomes are possible due to specific factors and mechanisms that function in synchrony. The mitotic promoting factors, the "structural maintenance of chromosomes" proteins, and proteins associated with cytoskeletal and nucleoskeletal elements have specific roles in structural modeling and functioning of DNA. It is of interest that the DNA decondensation cycle opposes the DNA condensation cycle. However, it is not clear if the factors and mechanisms involved in the DNA decondensation cycle are exactly opposite to the DNA condensation cycle. Also interesting is that chromosome-specific chromatin is positioned in the interphase nucleus in specific "territories" or "niches," a phenomenon similar to the exactly positioned genes at specific locations on a fully condensed chromosome. We review the factors and mechanisms in remodeling chromatin, maintaining structural integrity at each organizational level, and impact of this structural remodeling on functions of the genetic material.

Bleomycin-induced genotoxicity in vitro in human peripheral blood lymphocytes evidenced as complex chromosome- and chromatid-type aberrations.

Bleomycin is a chemotherapeutic and a radiomimetic drug which induces single and double-strand breaks in DNA by forming free radicals. We demonstrate in this study the capacity of bleomycin in inducing complex chromosome- and chromatid-type aberrations. Human peripheral blood was exposed to different concentrations of bleomycin (0, 10, 20, 30 and 40 µg/mL) and the aberrations induced were studied. The chromosomal-type aberrations studied were dicentrics, tricentrics, tetracentrics, centric rings and acentric fragments. The chromatid-type aberrations studied were double minutes, terminal lesions and terminal deletions. Though the overall trends that we obtained in the dose-dependent mitotic index and the chromosome- and chromatid-type aberrations conform to the reported literature, we could observe enhanced numbers and the types of such damages in this study. We could notice that chromosome-type aberrations were more than the chromatid-type aberrations. The enhanced numbers and the types of aberrations induced pave way for enhancing the sensitivity of genotoxic assays. Also, with more numbers and type of aberrations available, it would be useful to study the mechanisms of genotoxicity of drugs and in understanding phenomena such as "tolerance induction" to chronic exposure to such mutagens.

Cytokine profiling of MCF-7 cell line 2D, progressive 3D, and 3D revert cultures.

Cancer cytokines are known to mediate several complex cancer cell physiologies. Also, cancer cells themselves are known to secrete cytokines whose expressions and net inducible results are controlled by a variety of factors. We profiled a few cytokines secreted by 2D, 3D aggregates, and the 3DRs of MCF-7 cell line at various time points. Several cytokines were seen more expressed by 3D cultures on the 4th day and IL-10 peaked on the day 1 of 3D cultures while TNF-α level peaked on the 7th day. α-Defensin, SDF-7, and TGF-ß also showed markedly higher levels. There was a reduced expression of IL-6 and IL-17 by the 3DRs. TGF-ß did not show much change among the 2D, progressive 3D, and 3DR cultures. Utilizing 3DRs as study material will be a significant extension of the ways cells lines can be used for research.

Single-nucleotide polymorphism of INS, INSR, IRS1, IRS2, PPAR-G and CAPN10 genes in the pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common and a complex female endocrine disorder, and is one of the leading cause of female infertility. Here, we aimed to investigate the association of single-nucleotide polymorphism of INS, INSR, IRS1, IRS2, PPAR-G and CAPN10 gene in the pathogenesis of PCOS. A hospital-based, observational case-control study was carried on 169 PCOS and 169 control women in the southern region of India. Genotype was carried out by real-time polymerase chain reaction. A chi-square (χ2) test was performed and the genotypes were verified to comply with the Hardy- Weinberg equilibrium. Odds ratio and 95% confidence interval were calculated to assess the relative risk. Comparison of clinical characteristics of women with PCOS and controls reveal an increase in body mass index (BMI), luteinizing hormone / follicle stimulating hormone (LH/FSH) ratio, glucose levels, insulin, testosterone, hirsutism and antral follicular count in PCOS women. The variant rs1801278 (P = 0.002; OR = 2.88; 95% CI = 1.43, 5.80) show an association with PCOS. In the genotypic (P = 0.0002) and allelic models (P = 0.000), significance persisted even after Bonferroni correction. The genotypes of SNPs strongly influence BMI, LH, LH/FSH ratio, ovarian volume and antral follicular count in PCOS women. The study results were suggestive of a positive association between Gly972Arg of IRS1 and PCOS in the south Indian population, while INS, IRS2, PPAR-G and CAPN10 failed to show any association with PCOS in our studied population. Further studies focussing the role of IRS1 are warranted to delineate its implication towards PCOS.

Cancer Cytokines and the Relevance of 3D Cultures for Studying Those Implicated in Human Cancers.

Cancers are complex conditions and involve several factors for oncogenesis and progression. Of the various factors influencing the physiology of cancers, cytokines are known to play significant roles as mediators of functions. Intricate cytokine networks have been identified in cancers and interest in cytokines associated with cancers has been gaining ground. Of late, some of these cytokines are even identified as potential targets for cancer therapy apart from a few others such as IL-6 being identified as markers for disease prognosis. Of the major contributors to cancer research, cancer cell lines occupy the top slot as the most widely used material in vitro. In vitro cell cultures have seen significant evolution by the introduction of 3-dimensional (3D) culture systems. 3D cell cultures are now widely accepted as excellent material for cancer research which surpass the traditional monolayer cultures. Cancer research has benefited from 3D cell cultures for understanding the various hallmarks of cancers. However, the potential of these culture systems are still unexploited for cancer cytokine research compared to the other aspects of cancers such as gene expression changes, drug-induced toxicity, morphology, angiogenesis, and invasion. Considering the importance of cancer cytokines, 3D cell cultures can be better utilized in understanding their roles and functions. Some of the possibilities where 3D cell cultures can contribute to cancer cytokine research arise from the distinct morphology of the tumor spheroids, the extracellular matrix (ECM), and the spontaneous occurrence of nutrient and oxygen gradients. Also, the 3D culture models enable one to co-culture different types of cells as a simulation of in vivo conditions, enhancing their utility to study cancer cytokines. We review here the cancer associated cytokines and the contributions of 3D cancer cell cultures for studying cancer cytokines. J. Cell. Biochem. 118: 2544-2558, 2017. © 2017 Wiley Periodicals, Inc.

Genes Associated with Human Cancers: Their Expressions, Features, Functions, and Significance.

Various types of cancer continue to be subjects of intense research because of the impact of these diseases and their socioeconomic implications. Also, the complexity involved in the pathogenesis, nature of the triggers, and the progression of cancers is intriguing. An important aspect of cancers is the genetics involved, and studies involving cancer genes contributed immensely in not only understanding cancers better, but also for obtaining useful markers and therapy targets. We review the salient features, functions, and changes in gene expression for 103 carcinoma genes, 20 sarcoma genes, and 36 lymphoma genes. Apart from the three major levels of cancer type, we discuss the implications of altered gene expression at the tissue level as well. The possible uses of these gene functions and expression changes for diagnostic, prognostic, and therapeutic applications are presented. Also, the 159 genes are assessed for their involvement in more than a single cancer and tissue type. Only the p53 gene is commonly implicated in carcinomas, sarcoma and lymphomas. The CHEK2 and ERBB2 (HER2) genes are commonly found to be associated with carcinomas and sarcomas, whereas the MDM2, MSH2, and MSH6 genes are commonly implicated among carcinomas and lymphomas.

Receptors and signaling mechanisms for B-lymphocyte activation, proliferation and differentiation--insights from both in vivo and in vitro approaches.

During the last three decades, a number of B-lymphocyte specific surface antigens have been defined some of which may also show activation/differentiation specific expression. Here, we review the various signaling events and the receptor-ligand interactions for B-cell development, activation and differentiation. Our discussion and presentation include reviewing the in vivo and in vitro mechanisms. Focus is on the experiments that give us valuable insights into the B cell signaling mechanisms in vitro. Three significant pathways in B-cell development - c-Kit, FLT-3 and IL-7 signaling pathways are elucidated upon. Both antigen dependent and antigen independent mechanisms of B cell stimulation are also reviewed.

Generation of monoclonal antibodies to mitotic and interphase cytosolic proteins of Chinese hamster ovary (CHO) cells.

Mitotic proteins are well characterized and their cell cycle regulation roles studied extensively. Specific mitotic proteins can be key targets for controlling de regulated cell cycles. In the present study, cytosolic proteins of CHO (Chinese Hamster Ovary) cells were isolated and used for generating a range of monoclonal antibodies. Of the two antigenic doses utilized, 20 microg and 15 microg doses gave fusion efficiencies of 52.1% and 31.5% respectively. The specific efficiencies were found to be 24.5% for the 20 microg antigenic dose and was 20.45% for the 15 microg dose. Further screening showed 20 MAbs to common mitotic and interphase proteins, 5 specific to unique mitotic proteins and 3 to unique interphase proteins. MPFs do not exhibit species barriers and induce chromatin condensation and act as M- phase check point control molecules. Monoclonal antibodies to specific mitotic proteins can be very useful for various applications such as imaging tools, as possible mitotic inhibitors and also for affinity purification of specific proteins of interest. Antibodies specific to common mitotic and interphase proteins can also be of similar importance. Whole extract was used as immunogen also to present the mitotic proteins much better than isolated proteins.

Immunizations of human lymphocytes in vitro with a T-dependent antigen towards human monoclonal antibody production.

Human monoclonal antibodies have a plethora of applications, justifying the time and effort towards development of techniques for their efficient production. Attempts at establishing efficient reproducible techniques for activation of lymphocytes in culture have met with moderate success. In this study, human lymphocytes from peripheral blood and bone marrow were immunized in vitro with a T-dependent antigen--bovine gamma globulin. Whole blood, bone marrow and separated mononuclear peripheral blood cells were studied for the potential antibody secretory capabilities. The culture conditions included supplementation with heat treated autologous serum, spent medium from U-266 myeloma cell culture, which is known to contain B cell differentiation factors, and varied antigenic concentrations along with exposure duration. Although there was no appreciable difference in response between whole peripheral blood and whole bone marrow, there is a much better response when compared to isolated cell cultures; especially when culture conditions include antigenic withdrawal and supplementation with conditioned medium. However, lower antigenic concentration was required for whole bone marrow cultures. With optimal in vitro conditions for antigenic stimulation standardized, several options are available for the immortalization of such activated cells to obtain stable human hybridomas of interest.