Jana Karaskova

Immortal Human Pancreatic Duct Epithelial Cell Lines with Near Normal Genotype and Phenotype

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Spectral karyotyping identifies recurrent complex rearrangements of chromosomes 8, 17, and 20 in osteosarcomas.

Conventional cytogenetic studies have shown that osteosarcomas (OSs) are often highly aneuploid, with a large number of both structural and numerical chromosomal alterations. To investigate the complexity of OS karyotypes in detail, we applied spectral karyotyping (SKY) to a series of 14 primary OS tumors and four established OS cell lines. A total of 531 rearrangements were identified by SKY, of which 300 breakpoints could be assigned to a specific chromosome band. There was an average of 38.5 breakpoints identified by SKY per primary tumor. Chromosome 20 was involved in a disproportionately high number of structural rearrangements, with 38 different aberrations being detected. Chromosomal rearrangements between chromosomes 20 and 8 were evident in four tumors. FISH analysis using a 20q13 subtelomeric probe identified frequent involvement of 20q in complex structural rearrangements of OS cell lines. Characterization of the structural aberrations of chromosomes 8 and 17 by use of SKY demonstrated frequent duplication or partial gains of chromosome bands 8q23–24 and 17p11–13. Other chromosomes frequently involved in structural alteration were chromosomes 1 (47 rearrangements) and 6 (38 rearrangements). Centromeric rearrangements often involving chromosomes 1, 6, 13, 14, 17, and 20 were present. Four of the 14 primary OS tumors were characterized by nonclonal changes that included both structural and numerical alterations. In summary, OS tumors have a very high frequency of structural and numerical alterations, compounded by gross changes in ploidy. This intrinsic karyotype instability leads to a diversity of rearrangements and the acquisition of composite chromosomal rearrangements, with the highest frequency of alteration leading to gain of 8q23–24 and 17p11–13 and rearrangement of 20q. These findings suggest that specific sequences mapping to these chromosomal regions will likely have a role in the development and progression of OS.

The RAG-1/2 endonuclease causes genomic instability and controls CNS complications of lymphoblastic leukemia in p53/Prkdc-deficient mice

Double-strand DNA breaks (DSB) induce chromosomal translocations and gene amplification in cell culture, but mechanisms by which DSB cause genomic instability in vivo are poorly understood. We show that RAG-1/2-induced DSB cause IgH/c-Myc translocations in leukemic pro-B cells from p53/Prkdc-deficient mice. Strikingly, these translocations were complex, clonally heterogeneous and amplified. We observed reiterated IgH/c-Myc fusions on dicentric chromosomes, suggesting that amplification occurred by repeated cycles of bridge, breakage and fusion. Leukemogenesis was not mitigated in RAG-2/p53/Prkdc-deficient mice, but leukemic pro-B cells lacked IgH/c-Myc translocations. Thus, global genomic instability conferred by p53/Prkdc disruption efficiently transforms pro-B cells lacking RAG-1/2-induced DSB. Unexpectedly, RAG-2/p53/Prkdc-deficient mice also developed leptomeningeal leukemia, providing a novel spontaneous model for this frequent complication of human lymphoblastic malignancies.

An integrated mBAND and submegabase resolution tiling set (SMRT) CGH array analysis of focal amplification, microdeletions, and ladder structures consistent with breakage-fusion-bridge cycle events in osteosarcoma.

Osteosarcoma (OS) is characterized by chromosomal instability and high‐copy‐number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well‐established mechanism of genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high‐resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB—dicentric chromosomes, inverted duplications, and intra‐ and interchromosomal amplifications—were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG‐63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21–p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.

Brca1 required for T cell lineage development but not TCR loci rearrangement.

Brca1 (breast cancer1, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell–specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1−/− mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53−/− backgrounds, completely restored survival and development of Brca1−/− thymocytes; peripheral T cell numbers were not totally restored in Brca1−/− p53−/− mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1−/− thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model.

Cytotoxic drug treatment of neuroblastoma often leads to the development of drug resistance and may be associated with increased malignancy. To study the effects of long‐term cytotoxic treatment on malignant properties of tumor cells, we established 2 neuroblastoma cell sublines resistant to vincristine (VCR) and doxorubicin (DOX). Both established cell lines (UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100) were highly resistant to VCR, DOX and vice‐versa but retained their sensitivity to cisplatin. UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 expressed significant amounts of P‐glycoprotein, while parental cells were P‐glycoprotein negative. GD2 expression was upregulated, whereas NCAM expression was decreased in both resistant cells. Spectral karyotype (SKY) analysis revealed complex aberrant karyotypes in all cell lines and additional acquired karyotype changes in both resistant cells. All cell lines harbored high levels of N‐myc amplification. Compared to parental cells, UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 exhibited more than 2‐fold increase in clonal growth in vitro, accelerated adhesion and transendothelial penetration and higher tumorigenicity in vivo. We conclude that development of drug resistance and acquisition of certain karyotypic alterations is associated with an increase of additional malignant properties that may contribute to the poor prognosis in advanced forms of NB. The 2 novel neuroblastoma cell sublines also provide useful models for the study of drug resistance in aggressive forms of neuroblastoma.

Selection for transgene homozygosity in embryonic stem cells results in extensive loss of heterozygosity

Embryonic stem cells offer unprecedented opportunities for random or targeted genome alterations in the mouse. We present here an efficient strategy to create chromosome-specific loss of heterozygosity in embryonic stem cells. The combination of this method with genome-wide mutagenesis in ES cells (using chemical mutagens or gene-trap vectors) opens up the possibility for in vitro or in vivo functional screening of recessive mutations in the mouse.

Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer cell lines by sequential Giemsa banding and spectral karyotyping

AbstractBackground: Currently, prostate cancer (CaP) cytogenetics is not well defined, largely because of technical difficulties in obtaining primary tumor metaphases. Methods and Results: We examined three CaP cell lines (LNCaP, DU145, PC-3) using sequential Giemsa banding and spectral karyotyping (SKY) to search for a common structural aberration or translocation breakpoint. No consistent rearrangement common to all three cell lines was detected. A clustering of centromeric translocation breakpoints was detected in chromosomes 4, 5, 6, 8, 11, 12, 14, and 15 in DU145 and PC-3. Both these lines were found to have karyotypes with a greater level of complexity than LNCaP. Conclusion: The large number of structural aberrations present in DU145 and PC-3 implicate an underlying chromosomal instability and subsequent accumulation of cytogenetic alterations that confer a selective growth advantage. The high frequency of centromeric rearrangements in these lines indicates a potential role for mitotic irregularities associated with the centromere in CaP tumorigenesis.

Caspase-8 is essential for maintaining chromosomal stability and suppressing B-cell lymphomagenesis

In addition to its proapoptotic function, caspase-8 is also important for several other processes, including suppressing necroptosis, cell migration, and immune cell survival. In the present study, we report that the loss of caspase-8 in B lymphocytes leads to B-cell malignancies and that the risk for these tumors is further enhanced in the absence of p53. We also report that deficiency of caspase-8 results in impaired cytokinesis and that casp8(-/-) lymphomas display remarkably elevated levels of chromosomal aberrations. Our data support an important role for caspase-8 in the maintenance of genomic integrity and highlight its tumor-suppressive function.