Brian G. Stultz

Chromosomal stability of mesenchymal stromal cells during in vitro culture

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) are being investigated for use in cell therapy. The extensive in vitro expansion necessary to obtain sufficient cells for clinical use increases the risk that genetically abnormal cells will arise and be propagated during cell culture. Genetic abnormalities may lead to transformation and poor performance in clinical use, and are a critical safety concern for cell therapies using MSCs. METHODS We used spectral karyotyping (SKY) to investigate the genetic stability of human MSCs from ten donors during passaging. RESULTS Our data indicate that chromosomal abnormalities exist in MSCs at early passages and can be clonally propagated. The karyotypic abnormalities observed during our study diminished during passage. CONCLUSIONS Karyotyping of MSCs reveals characteristics which may be valuable in deciding the suitability of cells for further use. Karyotypic analysis is useful for monitoring the genetic stability of MSCs during expansion.

The Zic family member, odd-paired, regulates the Drosophila BMP, decapentaplegic, during adult head development.

The eye/antennal discs of Drosophila form most of the adult head capsule. We are analyzing the role of the BMP family member decapentaplegic (dpp) in the process of head formation, as we have identified a class of cis-regulatory dpp mutations (dpps-hc) that specifically disrupts expression in the lateral peripodial epithelium of eye/antennal discs and is required for ventral head formation. Here we describe the recovery of mutations in odd-paired (opa), a zinc finger transcription factor related to the vertebrate Zic family, as dominant enhancers of this dpp head mutation. A single loss-of-function opa allele in combination with a single copy of a dpps-hc produces defects in the ventral adult head. Furthermore, postembryonic loss of opa expression alone causes head defects identical to loss of dpps-hc/dpps-hc, and dpphc/+;opa/+ mutant combinations. opa is required for dpp expression in the lateral peripodial epithelium, but not other areas of the eye/antennal disc. Thus a pathway that includes opa and dpp expression in the peripodial epithelium is crucial to the formation of the ventral adult head. Zic proteins and members of the BMP pathway are crucial for vertebrate head development, as mutations in them are associated with midline defects of the head. The interaction of these genes in the morphogenesis of the fruitfly head suggests that the regulation of head formation may be conserved across metazoans.

Drosophila caliban, a nuclear export mediator, can function as a tumor suppressor in human lung cancer cells

We previously showed that the Drosophila DNA binding homeodomain of Prospero included a 28 amino-acid sequence (HDA) that functions as a nuclear export signal. We describe here the identification of a protein we named Caliban, which can directly interact with the HDA. Caliban is homologous to human Sdccag1, which has been implicated in colon and lung cancer. Here we show that Caliban and Sdccag1 are mediators of nuclear export in fly and human cells, as interference RNA abrogates export of EYFP-HDA in normal fly and human lung cells. Caliban functions as a bipartite mediator nuclear export as the carboxy terminus binds HDA and the amino terminus itself functions as an NES, which directly binds the NES receptor Exportin. Finally, while non-cancerous lung cells have functional Sdccag1, five human lung carcinoma cell lines do not, even though Exportin still functions in these cells. Expression of fly Caliban in these human lung cancer cells restores EYFP-HDA nuclear export, reduces a cells ability to form colonies on soft agar and reduces cell invasiveness. We suggest that Sdccag1 inactivation contributes to the transformed state of human lung cancer cells and that Caliban should be considered a candidate for use in lung cancer gene therapy.

Odd paired transcriptional activation of decapentaplegic in the Drosophila eye/antennal disc is cell autonomous but indirect

The gene odd paired (opa), a Drosophila homolog of the Zinc finger protein of the cerebellum (Zic) family of mammalian transcription factors, plays roles in embryonic segmentation and development of the adult head. We have determined the preferred DNA binding sequence of Opa by SELEX and shown that it is necessary and sufficient to activate transcription of reporter gene constructs under Opa control in transgenic flies. We have found a related sequence in the enhancer region of an opa-responsive gene, sloppy paired 1. This site also responds to Opa in reporter constructs in vivo. However, nucleotide alterations that abolish the ability of Opa to bind this site in vitro have no effect on the ability of Opa to activate expression from constructs bearing these mutations in vivo. These data suggest that while Opa can function in vivo as a sequence specific transcriptional regulator, it does not require DNA binding for transcriptional activation.

In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

BACKGROUND Mesenchymal stromal cells (MSCs) are being investigated as therapies for inflammatory diseases due to their immunosuppressive capacity. Interferon (IFN)-γ treatment primes MSC immunosuppression partially through induction of Indoleamine 2,3-dioxygenase (IDO1), which depletes tryptophan necessary to support proliferation of activated T cells. We investigated the role of histone modifications in the timing and maintenance of induced IDO1 expression in MSCs under clinical manufacturing conditions, such as cryopreservation. METHODS We used chromatin immunoprecipitation and quantitative polymerase chain reaction (PCR) to assay levels of transcriptionally permissive acetylated H3K9 and repressive trimethylated H3K9 histone modifications surrounding the transcriptional start site for IDO1, and reverse transcriptase PCR and immunoblotting to detect messenger RNA (mRNA) and protein. RESULTS MSCs derived from three donors approached maximum IDO1 mRNA levels following 24 hours of in vitro cytokine treatment. Induction of IDO1 expression correlated with increased acetylation of H3K9 concomitant with reduction of trimethylated H3K9 modifications at the promoter. Examination of two additional donors confirmed this result. While induced IDO1 levels decreased within 2 days after cytokine removal and freeze thawing, the activated chromatin state was maintained. Upon re-exposure to cytokines, previously primed MSCs accumulated near-maximum IDO1 mRNA levels within 4-8 h. DISCUSSION Our data indicate that in vitro priming of MSCs causes chromatin remodeling at the IDO1 promoter, that this alteration is maintained during processing commonly used to prepare MSCs for clinical use and that, once primed, MSCs are poised for IDO1 expression even in the absence of cytokines.

The tumor suppressor Caliban regulates DNA damage-induced apoptosis through p53-dependent and -independent activity.

We previously identified Caliban (Clbn) as the Drosophila homolog of human Serologically defined colon cancer antigen 1 gene and demonstrated that it could function as a tumor suppressor in human non-small-cell lung cancer (NSCLC) cells, although its mode of action was unknown. Herein, we identify roles for Clbn in DNA damage response. We generate clbn knockout flies using homologous recombination and demonstrate that they have a heightened sensitivity to irradiation. We show that normal Clbn function facilitates both p53-dependent and -independent DNA damage-induced apoptosis. Clbn coordinates different apoptosis pathways, showing a two-stage upregulation following DNA damage. Clbn has proapoptotic functions, working with both caspase and the proapoptotic gene Hid. Finally, ecotopic expression of clbn+ in NSCLC cells suppresses tumor formation in athymic nude mice. We conclude that Caliban is a regulator of DNA damage-induced apoptosis, functioning as a tumor suppressor in both p53-dependent and -independent pathways.

Dual Role of Jun N-terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.

Odd-Paired : The Drosophila Zic Gene

Zinc finger in the cerebellum (Zic) proteins are a family of transcription factors with multiple roles during development, particularly in neural tissues. The founding member of the Zic family is the Drosophila odd-paired (opa) gene. The Opa protein has a DNA binding domain containing five Cys2His2-type zinc fingers and has been shown to act as a sequence-specific DNA binding protein. Opa has significant homology to mammalian Zic1, Zic2, and Zic3 within the zinc finger domain and in two other conserved regions outside that domain. opa was initially identified as a pair-rule gene, part of the hierarchy of genes that establish the segmental body plan of the early Drosophila embryo. However, its wide expression pattern during embryogenesis indicates it plays additional roles. Embryos deficient in opa die before hatching with aberrant segmentation but also with defects in larval midgut formation. Post-embryonically, opa plays important roles in adult head development and circadian rhythm. Based on extensive neural expression, opa is predicted to be involved in many aspects of neural development and behavior, like other proteins of the Zic family. Consensus DNA binding sites have been identified for Opa and have been shown to activate transcription in vivo. However, there is evidence Opa may serve as a transcriptional regulator in the absence of direct DNA binding, as has been seen for other Zic proteins.

Jun N-terminal kinase signaling makes a face

ABSTRACT decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpps action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions.

Abstract 1108: The Drosophila Caliban suppresses tumor formation through both DNA damage repair and apoptosis

The tumor suppressor gene sdccag1 is down-regulated in human colon cancer cell lines, its Drosophila homolog caliban was screened using a functional HDA domain of homeobox gene prospero as bait. In our previous work, we had suggested Caliban is a bipartite nuclear export mediator, working through the Exportin pathway, and itself can shuttle between cytoplasm and nucleus. To study its tumor suppression functions, we generated knock-out Drosophila Melanogaster of caliban by gene targeting strategy. The caliban knock-out flies are viable and fertile, similar as tumor suppressor gene Dmp53 knock-out flies. While caliban null flies have retarded growth by three days, this delayed growth was rescued by Dmp53 mutation. The caliban flies are sensitive to irradiation, and its DNA damage sensitivity depends on p53. High dosage treatment with irradiation causes tumor formation in caliban null flies, while atm and caliban double mutant flies have autonomous tumor formation, which is similar as atm p53 double mutant. The tumor suppression function of caliban was also studied in an in vivo mouse model where lung tumor cells A549 were implanted subcutaneously into athymic nude mice, we observed the greatly reduced tumor size in caliban expressing A549 cells. These preliminary results suggest caliban may compensate with p53 to suppress the tumor predisposition through both DNA damage repair and apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1108.