Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms.

Cancer cells require telomere maintenance to enable uncontrolled growth. Most often telomerase is activated, although a subset of human cancers are telomerase-negative and depend on recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). ALT depends on proteins that are essential for homologous recombination, including BLM and the MRN complex, to extend telomeres. This study surveyed the requirement for requisite homologous recombination proteins, yet to be studied in human ALT cell lines, by protein depletion using RNA interference. Effects on ALT were evaluated by measuring C-circle abundance, a marker of ALT. Surprisingly, several proteins essential for homologous recombination, BARD1, BRCA2, and WRN, were dispensable for C-circle production, while PALB2 had varying effects on C-circles among ALT cell lines. Depletion of homologous recombination proteins BRCA1 and BLM, which have been previously studied in ALT, decreased C-circles in all ALT cell lines. Depletion of the non-homologous end joining proteins 53BP1 and LIG4 had no effect on C-circles in any ALT cell line. Proteins such as chromatin modifiers that recruit double-strand break proteins, RNF8 and RNF168, and other proteins loosely grouped into excision DNA repair processes, XPA, MSH2, and MPG, reduced C-circles in some ALT cell lines. MSH2 depletion also reduced recombination at telomeres as measured by intertelomeric exchanges. Collectively, the requirement for DNA repair proteins varied between the ALT cell lines compared. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways.

HECT-E3 ligase ETC-1 regulates securin and cyclin B1 cytoplasmic abundance to promote timely anaphase during meiosis in C. elegans.

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression. Securin protein levels change sharply during cell cycle progression, enabling its timely action. To understand the mechanism underlying the tightly regulated dynamics of securin, we analyzed the subcellular localization of the securin IFY-1 during C. elegans development. IFY-1 was highly expressed in the cytoplasm of germ cells. The cytoplasmic level of IFY-1 declined immediately following meiosis I division and remained low during meiosis II and following mitoses. We identified a C. elegans homolog of another type of E3 ligase, UBE3C, designated ETC-1, as a regulator of the cytoplasmic IFY-1 level. RNAi-mediated depletion of ETC-1 stabilized IFY-1 and CYB-1 (cyclin B1) in post-meiosis I embryos. ETC-1 knockdown in a reduced APC function background caused an embryonic lethal phenotype. In vitro, ETC-1 ubiquitylates IFY-1 and CYB-1 in the presence of the E2 enzyme UBC-18, which functions in pharyngeal development. Genetic analysis revealed that UBC-18 plays a distinct role together with ETC-1 in regulating the cytoplasmic level of IFY-1 during meiosis. Our study reports a novel mechanism, mediated by ETC-1, that co-operates with APC/C to maintain the meiotic arrest required for proper cell cycle timing during reproduction.

Identification and functional characterization of nuclear mortalin in human carcinogenesis.

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.

Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein.

CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

Five dysfunctional telomeres predict onset of senescence in human cells.

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.

MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3' untranslated region.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Tumor suppressor activity of miR-451: Identification of CARF as a new target.

microRNAs (miRs) have recently emerged as small non-coding regulators of gene expression. We performed a loss-of-function screening by recruiting retrovirus mediated arbitrary manipulation of genome coupled with escape of cells from 5-Aza-2'-deoxycytidine (5-Aza-dC)-induced senescence. miRNA pool from cells that emerged from 5-Aza-dC-induced senescence was subjected to miR-microarray analysis with respect to the untreated control. We identified miR-451 as one of the upregulated miRs and characterized its functional relevance to drug resistance, cell growth, tumor suppressor proteins p53 and pRb, and stress response. We report that miR-451 caused growth arrest in cells leading to their resistance to 5-Aza-dC-induced senescence. Decrease in cyclin D1, CDK4 and phosphorylated pRB supported the growth arrest in miR-451 transfected cells. We demonstrate that Collaborator of ARF (CARF) protein is a new target of miR-451 that intermediates its function in tumor suppressor and stress signaling.

Internalizing antibody-based targeted gene delivery for human cancer cells.

Gene therapy, a potential solution to hereditary and nonhereditary diseases, faces the challenges of safe and specific gene delivery. Cationic carrier molecules (e.g., liposome and polymers) that form noncovalent complexes with negatively charged DNA have been in use as nonviral gene delivery vectors. Although they tend to be relatively less efficient than viral systems, they have inherent advantages of flexibility and safety. Their derivatives, in conjugation with functional molecules such as peptides, proteins, growth factors, and antibodies, have been focused on to generate nanocarriers with low toxicity, high stability, high efficiency, and cell-specific targeting features. Here we describe internalizing polyclonal and monoclonal antibodies against a stress chaperone, mortalin/mtHsp70. We demonstrate that these internalizing anti-mortalin antibodies (i-mot Ab) could be employed for (1) internalization of nanoparticles (quantum dots, Qdots) and the generation of illuminating cells and (2) gene delivery. By using cancer and normal human cells in parallel, we further demonstrate that gene delivery can be specifically enhanced in human cancer cells if cationic polymer polyethylenimine (PEI) and i-mot Ab complex are used and may provide a novel cancer-targeting nanocarrier.

Quantum dot-based mortalin staining as a visual assay for detection of induced senescence in cancer cells.

Quantum dots (QDs) are fluorescent nanocrystals that are emerging as fine alternatives to the conventional organic dyes. They have several advantages including greater photostability and a wider range of excitation-emission wavelengths. By using mortalin staining as a model, we initially demonstrated that the QDs are more stable and provide better resolution in protein imaging in fixed cells. With the help of an internalizing antibody, we generated internalizing QD (i-QD) and demonstrated its inertness to cell replication, structure, and viability. Based on the superior resolution, stability and inertness, we propose the use of QD staining of mortalin as a cell-based visual assay to screen for senescence-inducing drugs, proteins, and siRNAs.

Withaferin-A kills cancer cells with and without telomerase: chemical, computational and experimental evidences.

Maintenance of telomere length is the most consistent attribute of cancer cells. Tightly connected to their capacity to overcome replicative mortality, it is achieved either by activation of telomerase or an Alternative mechanism of Lengthening of Telomeres (ALT). Disruption of either of these mechanisms has been shown to induce DNA damage signalling leading to senescence or apoptosis. Telomerase inhibitors are considered as potential anticancer drugs but are ineffective for ALT cancers (~15% of all cancers). Withaferin-A (Wi-A), a major constituent of the medicinal plant, Withania somnifera (Ashwagandha), has been shown to exert anti-tumour activity. However, its effect on either telomerase or ALT mechanisms has not been investigated. Here, by using isogenic cancer cells with/without telomerase, we found that Wi-A caused stronger cytotoxicity to ALT cells. It was associated with inhibition of ALT-associated promyelocytic leukemia nuclear bodies, an established marker of ALT. Comparative analyses of telomerase positive and ALT cells revealed that Wi-A caused stronger telomere dysfunction and upregulation of DNA damage response in ALT cells. Molecular computational and experimental analyses revealed that Wi-A led to Myc-Mad mediated transcriptional suppression of NBS-1, an MRN complex protein that is an essential component of the ALT mechanism. The results suggest that Wi-A could be a new candidate drug for ALT cancers.

Quantum dot-based protein imaging and functional significance of two mitochondrial chaperones in cellular senescence and carcinogenesis.

Mortalin/mtHSP70 and HSP60 are heat-shock proteins that reside in multiple subcellular compartments, mitochondria being the dominant compartment. We present here biochemical evidence for their in vivo and in vitro interactions. By the use of quantum dots (powerful tools used for simultaneous imaging of multiple proteins), we visualized minute differences in the subcellular niche of these two proteins in normal and cancer cells. Knockdown of either of these two by shRNA expression plasmids caused growth arrest of osteosarcoma cells. However, interestingly, whereas an overexpression of mortalin extended in vitro life span of normal fibroblasts (TIG-1), overexpression of HSP60 was neutral. We demonstrate the minute differences in subcellular distribution of mortalin and HSP60, their involvement in tumorigenesis, and functional distinction in pathways involved in senescence.

Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60.

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF.

Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2'-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16(INK4A), pRB, HDM2 and p21(WAF1).

Stress chaperone mortalin contributes to epithelial-mesenchymal transition and cancer metastasis.

Mortalin/mthsp70 (HSPA9) is a stress chaperone enriched in many cancers that has been implicated in carcinogenesis by promoting cell proliferation and survival. In the present study, we examined the clinical relevance of mortalin upregulation in carcinogenesis. Consistent with high mortalin expression in various human tumors and cell lines, we found that mortalin overexpression increased the migration and invasiveness of breast cancer cells. Expression analyses revealed that proteins involved in focal adhesion, PI3K-Akt and JAK-STAT signaling, all known to play key roles in cell migration and epithelial-to-mesenchymal transition (EMT), were upregulated in mortalin-expressing cancer cells. We further determined that expression levels of the mesenchymal markers vimentin (VIM), fibronectin (FN1), ß-catenin (CTNNB1), CK14 (KRT14) and hnRNP-K were also increased upon mortalin overexpression, whereas the epithelial markers E-cadherin (CDH1), CK8 (KRT8), and CK18 (KRT18) were downregulated. Furthermore, shRNA-mediated and pharmacological inhibition of mortalin suppressed the migration and invasive capacity of cancer cells and was associated with a diminished EMT gene signature. Taken together, these findings support a role for mortalin in the induction of EMT, prompting further investigation of its therapeutic value in metastatic disease models.

Targeting of DNA Damage Signaling Pathway Induced Senescence and Reduced Migration of Cancer cells.

The heat shock 70 family protein, mortalin, has pancytoplasmic distribution pattern in normal and perinuclear in cancer human cells. Cancer cells when induced to senesce by either chemicals or stress showed shift in mortalin staining pattern from perinuclear to pancytoplasmic type. Using such shift in mortalin staining as a reporter, we screened human shRNA library and identified nine senescence-inducing siRNA candidates. An independent Comparative Genomic Hybridization analysis of 35 breast cancer cell lines revealed that five (NBS1, BRCA1, TIN2, MRE11A, and KPNA2) of the nine genes located on chromosome regions identified as the gain of locus in more than 80% cell lines. By gene-specific PCR, these five genes were found to be frequently amplified in cancer cell lines. Bioinformatics revealed that the identified targets were connected to MRN (MRE11-RAD50-NBS1) complex, the DNA damage-sensing complex. We demonstrate that the identified shRNAs triggered DNA damage response and induced the expression of tumor suppressor protein p16(INK4A) causing growth arrest of cancer cells. Furthermore, cells showed decreased migration, mediated by decrease in matrix metalloproteases. Taken together, we demonstrate that the MRN complex is a potential target of cancer cell proliferation and migration, and staining pattern of mortalin could serve as an assay to identify senescence-inducing/anticancer reagents.

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates.

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their i) broad absorption spectra allowing visualization with single light source, ii) exceptional photo-stability allowing long term studies and iii) narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.

Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells.

Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom's syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits.

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i-QDs) in the reparative tissues, following the allogeneic transplantation of three-dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein-70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i-QDs were added to the culture medium for 24 h. Scaffold-free cartilaginous aggregates formed from i-QD-labelled MSCs (i-MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin-O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti-mortalin antibody-conjugated i-QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i-MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects.

CARF Is a vital dual regulator of cellular senescence and apoptosis.

The tumor suppressor protein, p53, is central to the pathways that monitor the stress, DNA damage repair, cell cycle, aging, and cancer. Highly complex p53 networks involving its upstream sensors and regulators, downstream effectors and regulatory feedback loops have been identified. CARF (Collaborator of ARF) was shown to enhance ARF-dependent and -independent wild-type p53 function. Here we report that (i) CARF overexpression causes premature senescence of human fibroblasts, (ii) it is vital for replicative and stress-induced senescence, and (iii) the lack of CARF function causes aneuploidy and apoptosis. We provide evidence that CARF plays a dual role in regulating p53-mediated senescence and apoptosis, the two major tumor suppressor mechanisms.