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Human breast cancer is characterized by a high degree of inter-patients heterogeneity in terms of histology, genomic alterations, gene expression patterns, and metastatic behavior, which deeply influences individual prognosis and treatment response. The main cause of mortality in breast cancer is the therapy-resistant metastatic disease, which sets the priority for novel treatment strategies for these patients. In the present study, we demonstrate that Patient Derived Xenografts (PDXs) that were obtained from metastatic and therapy-resistant breast cancer samples recapitulate the wide spectrum of the disease in terms of histologic subtypes and mutational profiles, as evaluated by whole exome sequencing. We have integrated genomic and transcriptomic data to identify oncogenic and actionable pathways in each PDX. By taking advantage of primary short-term in vitro cultures from PDX tumors, we showed their resistance to standard chemotherapy (Paclitaxel), as seen in the patients. Moreover, we selected targeting drugs and analyzed PDX sensitivity to single agents or to combination of targeted and standard therapy on the basis of PDX-specific genomic or transcriptomic alterations. Our data demonstrate that PDXs represent a suitable model to test new targeting drugs or drug combinations and to prioritize personalized therapeutic regimens for pre-clinal and clinical tests.

Karyotypic destabilization in cells of Chinese hamster fibroblasts CHL V-79 RJK with ectopically overexpressed antiapoptotical human bcl-2 gene from pSFFV-bcl-2 vector has been analysed. Analysis of G-banded metaphase chromosomes from 4 clones with different levels of bcl-2 expression revealed an increased level of chromosomal instability in bcl-2-transfected cells. Besides, an increased percentage of aneu- and polyploid cells and high level of cells with different chromosomal aberrations was observed. The degree of karyotypic instability positively correlated with the level of bcl-2 expression in bcl-2-transfected cells. Cells of a clone with the highest bcl-2 expression at the 13th passage of cultivation displayed an almost 100% polyploidization and the presence of specific aberrations and a tricentric marker chromosome. Selection of cells with non-random specific chromosome changes was observed in pSFFV-bcl-2-transfected CHL V-79 RJK cells in the process of their long-term cultivation. By contrast, cells of the parental cell line, as well as the control pSFFV-neo transfectants, displayed a stable karyotype throughout the long period of cultivation. It is important that the presence of morphological markers of gene amplifications--DOO, DM, MH--was observed in bcl-2-transfected cells. These findings suggest that the overexpression of antiapoptotic human bcl-2 gene may result in destabilization of the karyotype structure in cells of Chinese hamster fibroblasts CHL V-79 RJK. The character and level of destabilization correlate with the level of ectopic overexpression of this gene.

Mechanisms of drug-resistance in two K562 cell lines selected for adriamycin and etoposide resistance (K562-ADR and K562-VP16, respectively) were studied. In K562-ADR cells, overexpression of mdr 1 gene and two-fold reduction of topoisomerase II alpha mRNA content were found, while topoisomerase II beta expression remained unchanged, compared to the parental cell line. Antiapoptotic bcl-2 mRNA level was four-fold decreased in K562-ADR cells, while the expression of other members of bcl-2 family was unaffected. In K562-VP16 cells five-fold reduction of topoisomerase II alpha expression was found with the absence of mdr 1 gene overexpression. The expression of antiapoptotic bcl-2 and proapoptotic bax genes was reduced in K562-VP16 cell line, while the content of bcl-2 mRNA was increased. Cytogenetic analysis of K562-VP16 cells revealed morphological changes in their cell karyotype and susceptibility of these cells to spontaneous polyploidization. Possible effects of etoposite on mitotic control in K562-VP16 cells are discussed.

Chinese hamster fibroblasts CHL V-79 RJK were subjected to multistep selection in the presence of etoposide, known as an inhibitor of topoisomerase II and inductor of apoptosis. The karyotype of cells stably resistant to etoposide was analysed at progressive stages of selection using G-type staining of metaphase chromosomes. Multiple changes in the karyotype of resistant cells were observed at an early stage of selection (0.2 mg/ml of etoposide) and included: random chromosome breaks leading to formation of new chromosome markers, high frequency of aneuploid and polyploid cells, morphological instability and extracopy of q-shoulder of chromosome 1. On advanced stages of selection we observed an increased frequency of specific minute chromosome and the appearance of chromosomes with homogeneously or differentially stained regions (HSR and DSR). These data suggest that different mechanisms may be involved in developing cellular resistance to etoposide at progressive stages of selection.

The endonuclease activity of two drug-sensitive and drug-resistant mouse myeloma cell lines during cytotoxic drug-induced apoptosis was studied. It was shown that internucleosomal fragmentation of DNA in drug-sensitive line sp2/0, undergoing apoptosis in the presence of adriamycin and colchicine, was not dependent on intracellular calcium content and was associated with activation of both Ca(2+)-Mg(2+)-dependent and acidic cation-independent endonucleases. In contrast, in multidrug resistant spEBR-5 cells, treated with the same drugs, only Ca(2+)-Mg(2+)-dependent endonuclease activity was detected. These data suggest that the differences in the pattern of endonuclease activity revealed in these cells are linked to drug-resistant phenotype and do not depend on the apoptosis-inducing agent used.

Target cells, K562 strain and its sublines characterized by multiple drug resistance (MDR) do not differ in their susceptibility to human natural killer cells (NK) but MDR cells are more susceptible to cytotoxic action of lymphokine-activated cells (LAC) and to NK cells in the presence of a selective agent adriamycin. Target cells death is characterized by fragmentation of nuclear DNA. It has been established that K562 thermotolerant subclone is more resistant to NK and LAC than other clones. Heat shock protein synthesis may have a protective impact in target cells death during interaction with NK and LAC cells.

A dose-dependent stimulation of immunoglobulin synthesis by Spebr-5 cells in the presence of adriamycin is shown. This effect is significantly blocked in the presence of iron-chelating agent 2,2-bipyridine. Moreover, low doses of H2O2 and heat shock also caused enhancement of immunoglobulin synthesis. These data suggest the involvement of free radical processes in the adriamycin induced stimulation of immunoglobulin synthesis.

Effects of cytotoxic drugs, doxorubicin (adriamycin) and ethidium bromide, on immunoglobulin production have been studied in B-cell hybridomas, myelomas and human lymphocytes. Doxorubicin, and in a lesser extent ethidium bromide, are shown to enhance immunoglobulin production both in drug-resistant and drug-sensitive cell lines. Enhancement of immunoglobulin production is accompanied with that of the number of antibody forming cells and with immunoglobulin synthesis in these cells.

The binding of adriamycin and ethidium bromide coupled to sepharose beads with intracellular and serum immunoglobulins is detected. These drugs do not bind with Fab and Fc fragments of immunoglobulin. The binding of ethidium bromide to immunoglobulins is competitive. Possible adaptive features of this phenomenon are discussed.