Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML.

Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress-associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).

Analysing kinetochore function in human cells: spindle checkpoint and chromosome congression.

During cell division microtubules of the mitotic spindle segregate the duplicated chromosomes into the two daughter cells. Chromosome-microtubule attachment is mediated by kinetochores, multiprotein complexes assembled on specialized regions of the DNA. Kinetochores modulate microtubule dynamics to generate the forces necessary to power chromosome movement and regulate the spindle checkpoint. Errors in kinetochore function can cause aneuploidy, a hallmark of 80% of solid tumors in humans, suggesting a fundamental link to tumorigenesis. Human kinetochores are complex protein machines with over 100 different proteins. Here we present fixed- and live-cell-based assays used to functionally categorize kinetochore proteins with regard to spindle checkpoint activity and kinetochore-microtubule attachment.

Characterization of {gamma}-aminobutyric acid type A receptor-associated protein, a novel tumor suppressor, showing reduced expression in breast cancer.

Frequent allelic loss of the chromosomal region 17p13 in breast cancer has suggested that more tumor suppressor genes, besides p53, are located in this region. By doing suppression subtractive hybridization to detect differentially expressed genes between the breast cancer cell line CAL51 and a nontumorigenic microcell hybrid CAL/17-1, we identified the gene for the gamma-aminobutyric acid type A (GABA(A)) receptor associated protein (GABARAP), located on 17p13.1. GABARAP displayed high expression levels in the microcell hybrid CAL/17-1 but only weak expression in CAL51 and other breast cancer cell lines tested. Furthermore, we observed large vesicles in CAL/17-1 by immunofluorescence staining, whereas no signal could be detected in the tumor cell line. GABARAP mRNA expression and protein expression were significantly down-regulated in invasive ductal and invasive lobular carcinomas compared with normal breast tissue measured by semiquantitative reverse transcription-PCR and immunohistochemistry, respectively. We assessed that neither mutations in the coding region of the gene nor hypermethylation of CpG islands in the promoter region are responsible for loss of gene expression in CAL51; however, 5-aza-2'-deoxycytidine treatment was effective in gene up-regulation, suggesting a methylation-dependent upstream effect. Stable transfection of GABARAP into CAL51 resulted in an increase of gene expression and remarkably influenced the ability of colony formation in soft agar and the growth rate in vitro and, moreover, suppressed the tumorigenicity of the cells in nude mice. In summary, our data suggest that GABARAP acts via a vesicle transport mechanism as a tumor suppressor in breast cancer.

Bub1 regulates chromosome segregation in a kinetochore-independent manner.

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458-476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.

Genetic background of different cancer cell lines influences the gene set involved in chromosome 8 mediated breast tumor suppression.

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.

Profile of differentially expressed genes after transfer of chromosome 17 into the breast cancer cell line CAL51.

Previous studies have shown that transfer of chromosome 17 suppresses the tumorigenic phenotype of the breast cancer cell line CAL51, suggesting the presence of putative tumor suppressor genes on this chromosome. Suppression subtractive hybridization and oligonucleotide microarray analyses were performed to identify differentially expressed genes in nontumorigenic microcell hybrids, CAL/17-1 and CAL/17-3, when compared with CAL51 cells. In total, 263 differentially expressed transcripts were associated with these phenotypes. Of these, a high percentage is involved in various biological processes associated with tumorigenesis, including DNA-dependent regulation of transcription, regulation of cell cycle, signal transduction, and cell proliferation. Microarray analysis of selected chromosome 17 genes in a series of 25 human primary breast tumors showed associations with clinicopathologic parameters of the tumors. Of these genes, TOB1 (transducer of ERBB2) was selected for further expression analysis. Using RT-PCR and immunohistochemical staining of tissue microarrays, we could reveal a differential mRNA and protein expression of TOB1 in the majority of breast tumors and lymph node metastases compared with normal breast tissues, indicating a potential role of this protein in breast tumorigenesis.