REGULATION OF GENE EXPRESSION, CELLULAR PROLIFERATION, AND DIFFERENTIATION IN MAMMALIAN DEVELOPMENT
, Head, Section on Developmental Genomics
Shao-Ming Wu, PhD, Staff Scientist
Maria Grekova, MD, PhD, Research Fellow
Tin-Lap Lee, PhD, Research Fellow
Vanessa Baxendale, MS, Research Associate
Xingli Meng, MD, PhD, Postdoctoral Fellow
Albert Hoi-Hung Cheung, MPhil, Graduate Student
Jessica Shookhoff, BS, Graduate Student
Elaina Mikhaylova, BS, Postbaccalaureate Fellow
Stephanie Peacock, BS, Postbaccalaureate Fellow
Lisa Ruszczyk, BS, Postbaccalaureate Fellow
Diana H. Taft, BS, Postbaccalaureate Fellow
Lisa Ruszczyk, BS, Summer Student
Morgan Ruszczyk, BS, Summer Student
Jonathan Tarn, Summer Student
Regulation of Gene Expression, Cellular Proliferation, and Differentiation in Mammalian Development
Identification and functional characterization of spermatogenesis stage–specific genes
At different stages of spermatogenesis, male germ cells evidence distinct morphological features or genetic markers that allow their easy identification and preparation. Based on expression analysis of mouse type A spermatogonia, pachytene spermatocytes, and round spermatids, we cloned several genes that are expressed at specific stages of spermatogenesis. They include mArd2, a novel mArd1 (Arrest Defective 1) homologue, that is testis-specific and expressed in meiotic and post-meiotic cells; mLin28, an evolutionarily conserved RNA-binding protein that is expressed in mitotic spermatogonia and implicated in temporal control of cellular development; and variants of Ddx3y, a Y-encoded gene expressed in spermatocytes. By performing in vitro protein pull-down and enzyme activity assays, we showed that mArd2 displays N-acetyltransferase activity. Starting from meiosis, mArd2 may therefore compensate for the loss of X-linked Ard1. Preliminary studies in a P19 embryonal carcinoma cell model revealed that Lin28 binds to specific sets of RNA transcripts that encode ribosomal proteins. This observation led us to hypothesize that Lin28 acts as an RNA chaperone and controls the availability of transcripts encoding products important for cell fate decisions.
More recently, based on the Serial Analysis of Gene Expression (SAGE) database that we generated, we cloned a novel testis-specific isoform of Cdc14A. This transcript, which we named sCdc14A, is expressed only in meiotic spermatocytes and post-meiotic spermatids; it has a very low level of expression in the ovary. When compared with sCdc14A, Cdc14A revealed a unique 3¢ UTR sequence despite a similar 5¢ UTR. The coding exons are slightly different, indicating splicing variation. The non–testis-specific human homologue of sCdc14A was shown to be an evolutionarily conserved, dual-specificity protein phosphatase that plays an important role in centrosome duplication and mitotic regulation. We raised antibodies against a polypeptide specific for sCdc14A. We will further characterize this protein and examine the regulatory mechanism of its expression.
Previous work showed that the GDNF receptor-a-1 (GFRA1) is specifically expressed in spermatogonial stem cells (SSCs) and is required for SSCs’ stem cell properties. To characterize the molecular phenotype of SSCs, we isolated GFRA1+ and GFRA1− spermatogonia from 6-day-old mice by using magnetic-activated cell sorting with an antibody to GFRA1 and compared the spermatogonias’ microarray-based expression profiles. We found that the expression of several genes was upregulated in GFRA1+ spermatogonia, with Csf1r the most overexpressed gene; the gene encodes the receptor for granulocyte-macrophage colony–stimulating factor (GM-CSF), which has a well-established role in hematopoietic stem cell function. Several chemokine ligands were also highly overexpressed in SSCs. Experiments are under way to dissect the role of chemokines in the stem cell properties of GFRA1+ spermatogonia.
Characterization of selected genetic processes in spermatogenesis
Using SAGE, we established an expression database of mouse male germ cells. Computational analyses had led to the identification of stage-specific pathways and promoter modules and the construction of biological networks associated with different stages of spermatogenesis. In addition, the analyses resulted in the identification of a large number of genes with stage-specific alternative splice variants. It has been suggested that the genetic process of alternative splicing is prominent during spermatogenesis. Several genes have been known to undergo alternative splicing, which confers novel activities to the variants. We initiated characterization of novel stage-specific variants of several genes, including heat shock protein 4 (Hspa4), H3 histone family 3B (H3f3b), and ubiquitin protein ligase E3A (Ube3a). We will use Hspa4 as a model to investigate the role of alternative splicing in stage-specific regulation of gene function and the impact of the splice variants’ biological activity. Oxidative stress induced Hspa4, which is critical for the survival and normal functioning of spermatozoa and for male fertility. We confirmed the presence of three distinctive transcripts of Hspa4 in type A spermatogonia, pachytene spermatocytes, and round spermatids. Further biochemical and functional studies are under way to characterize the gene’s regulatory mechanisms and biological functions.
Analysis of the germ cell SAGE database also revealed a relative paucity of antisense transcripts. We are particularly intrigued by the presence of antisense transcripts derived from pseudogenes. Among the 19 genes with antisense transcripts, four (Uba52, Ch10, Calm2, and Ubb) had antisense transcripts derived from their pseudogenes on different chromosomes. Apparently, these pseudogenes were derived from reverse transcripts of the respective parent genes and transposed to the intron of actively transcribed genes: the Uba52 pseudogene resides in the intron of Cbx1; the Calm2 pseudogene in the intron of Prkar2b; the Ch10 pseudogene in the intron of Sp3; and the Ubb pseudogene in the intron of Catsper2. More interestingly, the pseudogenes are antiparallel to their host genes. Thus, the antisense transcripts of the pseudogenes will be produced as processed introns of their respective host genes, raising the possibility that the two antiparallel transcription units interact through hybridization of the sense-antisense transcripts. Subsequent experiments confirmed the presence of native double-stranded RNA of the antiparallel genes Uba52-Cbx1, Ch10-Sp3, and Calm2-Prkar2b. We have not yet confirmed the presence of double-stranded RNA of Ubb-Catsper2. Using Uba52 and Cbx1 as a model, we will examine the relationship between the antiparallel gene pairs. The functional gene of Uba52 is on chromosome 8 while its pseudogene is embedded in the first intron of Cbx1on chromosome 11. Uba52, Cbx1, and the sense and antisense transcripts of the Uba52 pseudogene are expressed in the mouse kidney cell line CRL-6436, which we will use as a model for study.
Analyses of the antisense transcripts suggested the existence of RNA-dependent RNA polymerase (RdRP) activity in the mouse germ cells. We identified antisense transcripts complementary to several coding exons for Tcte3, Ldh3, and Calm2. We focused on the Calm2 antisense transcript, which was present in mouse testis and three mouse cell lines, namely, CRL-2576 (mouse spermatogonia cell line), CRL-1715 (mouse Sertoli cell line), and CRL-6436 (mouse kidney cell line). A knockdown experiment confirmed that the antisense transcript was a product of the sense transcript. Knocking down the sense transcript of Calm2 with siRNA demonstrated a reduction in the levels of both the sense and antisense transcripts, indicating that synthesis of the Calm2 antisense transcript was dependent on the sense transcript. Calm2 antisense was not synthesized starting from the 3¢ end of the sense mRNA. We defined the sequence representing the potential start site of the action of RdRP, generated a hybrid RNA containing this sequence ligated to EGFP on its 5¢ end, and introduced it into CRL-6436 cells. Orientation-specific RT-PCR showed production of an antisense RNA derived from hybrid RNA. The results provide further proof of the existence of RdRP activity in mammalian cells. Experiments to isolate and purify RdRP activity are under way.
Chan WY, Wu SM, Ruszczyk L, Law E, Lee TL, Baxendale V, Rennert OM. The complexity of antisense transcription revealed by the study of developing male germ cells. Genomics 2006;87:681-92.
Chan WY, Wu S, Ruszczyk LM, Lee T, Rennert OM. Antisense transcription in developing male germ cells. In: Lau YFC, Chan WY, eds. Y Chromosome and Male Germ Cell Biology in Health and Diseases. World Scientific Publishers, 2007;201-20.
Lee TL, Alba D, Wu SM, Baxendale V, Rennert OM, Chan WY. Application of transcriptional network analyses in mouse germ-cell transcriptomes. Genomics 2006;88:18-33.
Global expression mapping of developing male germ cells
Knowledge gained from the recently completed mammalian genomes revealed significant limitations of the current gene module algorithm and suggested that transcription units are more complicated than previously thought. We developed an algorithm and applied systems biology approaches to expand the capacity of SAGE, the sequence-based profiling technology. Application of this novel approach to developing male germ cells allowed a whole-genome view of antisense transcription, stage-specific alternative splicing, stage-specific regulatory circuitry, and promoter regulation and resulted in the identification of several novel transcripts and pseudogenes.
We leveraged our findings from SAGE by incorporating whole-genome 25 bp–resolution tiling expression arrays from Affymetrix®. With 45 million oligonucleotide probes and 35 bp probe spacing, we generated a high-definition transcriptome map of developing male germ cells with an unbiased and germ cell–specific whole-genome expression map. Preliminary data proved that, by combining it with the SAGE data set, the tiling platform is powerful and provides new insights into germ cell studies. We used pooled cDNA targets derived from poly A+–enriched RNA purified from type A spermatogonia, pachytene spermatocytes, and round spermatids to hybridize to tiling expression arrays; we next developed statistical models and bioinformatic algorithms for data analysis. We are currently optimizing our data analysis. Overall, we found that more than 45 percent of transcripts were not annotated; current annotation accounts for only about 30 percent of the data set, with the rest of the data set in the form of mostly expressed sequence tags (ESTs). We obtained reliable and reproducible results by using different reference genes known to be differentially expressed at different germ cell stages, such as the protamin gene family (Prm) members (Figure 11.1), meiosis-expressed gene (meig1), and spermatogonia-specific gene Lin28. We were able to identify antisense transcripts and pseudogenes revealed in our previous SAGE study and validated by RT-PCR and to confirm them in the tiling array experiment. Tiling array data provide a more comprehensive picture of the configuration of the transcript unit than with SAGE alone, allowing better gene module prediction and validation.
Figure 11.1
Expression pattern of Prm family members revealed by transcript tiling arrays. Spermatogenic cell types to which the tiling array patterns belong are indicated on the left; Spga = spermatogonia; Spcy = spermatocytes; Sptd = spermatids.
Initial analysis revealed expression of 4,421 gene modules that had not been previously annotated. Sequence analyses of these modules suggested that the predicted gene structure of nearly half of them needs improvement. Coupled with SAGE and CAGE (Cap Analysis Gene Expression) gene module mapping, tiling microarray analysis identified 482 new high-quality gene modules for the genome, representing an 11 percent increase in annotated protein-coding capacity. Previous annotation relied on ESTs or other cDNA sequences, alignment to protein sequences, comparative analysis of genomes, or de novo prediction programs that use statistical models to detect codons and conserved motifs for transcription initiation, polyadenylation, and splicing. These annotation approaches are deficient. The high-resolution detection of the transcript unit provided more reliable data in predicting splicing variants (Figure 11.2). We plan to apply the RACE-chip strategy to obtain full details of stage-specific variants or antisense transcripts. Further, to gain better insight into other potential regulatory mechanisms, we will analyze the tiling data with the Encyclopedia of DNA Elements (ENCODE) database. With this approach, we have already found a surprisingly large number of noncoding RNAs (ncRNAs) that give rise to unannotated transcripts or novel noncoding isoforms of protein-coding genes. We will continue to employ the tiling platform to study the potential of ncRNAs, such as miRNAs, as regulatory molecules during germ cell development. The results will be valuable in explaining regulation of the gene signatures at particular germ cell stages.
Figure 11.2
Combination of SAGE and CAGE with expression tiling array allowed identification of novel gene modules. Tag indicates SAGE tags. Different splicing variants at different stages of spermatogenesis are indicated. Spermatogenic cell types to which the tiling array patterns belong are indicated on the left.
Chan WY, Lee TL, Wu SM, Ruszczyk L, Alba D, Baxendale V, Rennert OM. Transcriptome analyses of male germ cells with serial analysis of gene expression (SAGE). Mol Cell Endocrinol 2006;250:8-19.
He Z, Chan WY, Dym M. Microarray technology offers a novel tool for the diagnosis of and identification of therapeutic targets for male infertility. Reproduction 2006;132:11-9.
Pang ALY, Johnson W, Dym M, Rennert OM, Chan WY. Expression profiling of purified male germ cells: stage specific expression patterns related to meiosis and post-meiotic development. Physiol Genomics 2006;24:75-85.
Structural and Biological Studies on Luteinizing Hormone/Chorionic Gonadotropin Receptor
Structural implications of disease-causing LH/CG-R mutations
LH/CG-R plays a central role in human male sexual development. Mutation of LH/CG-R results in abnormal production of testosterone and disorders of sexual development. While the role of LH/CG-R in transducing the signal of LH/CG binding is well established, the mechanism of its action is still not fully understood. We studied the molecular genetics of the LH/CG-R of a large number of patients with activating and inactivating mutations of the receptor. We observed that activating mutations of LH/CG-R lead to the development of familial male-limited precocious puberty (FMPP) and that inactivating mutations of the receptor lead to Leydig cell hypoplasia (LCH). About 90 percent of the FMPP patients in our study had an identifiable mutation of LH/CG-R. Mutation detection in LCH patients continues to be rather difficult. Nevertheless, studies on these naturally occurring mutations continue in order to help us understand the varied presentation of FMPP and LCH as well as the molecular mechanism of signal transduction of the receptor.
In a patient with LCH, we identified, by nucleotide sequencing of LH/CG-R, the novel heterozygous mutation A340T, which leads to the substitution of Phe for the conserved amino acid Ile-114. The mutation is located in the third leucine-rich repeat (LRR) in the ectodomain of LH/CG-R. In vitro expression studies demonstrated that the mutation results in reduced ligand binding and signal transduction of the receptor. Studies on LH/CG-R constructs, in which we substituted various amino acids for the conserved Ile-114, showed that receptor activity is sensitive to changes in the size, shape, and charge of the side chain. We constructed a homology model of the wild-type LH/CG-R ectodomain in order to illustrate the packing of conserved hydrophobic side chains in the protein core. Substitution of Ile-114 by Phe likely disrupted intermolecular contacts between hormone and receptor. This mutation might also affect an LH/CG-R–dimer interaction. Thus, the Ile114Phe mutation reduces ligand binding and signal transduction by LH/CG-R and is partially responsible for LCH in the patient.
Jeha GS, Lowenthal ED, Chan WY, Wu SM, Karaviti LP. Variable presentation of precocious puberty associated with the D564G mutation of the LHCGR gene in children with testotoxicosis. J Pediatr 2006;149:271-4.
Leung MYK, Steinbach P, Bear D, Baxendale V, Fechner PY, Rennert OM, Chan WY. Biological effect of a novel mutation in the third leucine-rich repeat of human luteinizing hormone receptor. Mol Endocrinol 2006;20:2493-503.
Function of hCG/LH and their receptor in the mammalian nervous system
Individuals with LH/CG-R carrying activating mutations develop FMPP and often exhibit behavioral problems, which may be related to the dysfunction of brain cells caused by the mutated receptor. Recent studies showed that hCG and its receptor LH/CG-R may have nongonadal functions that could be physiologically important. We found that, in the nervous system, LH/CG-R was expressed in the mammalian brain in a temporal and spatial pattern. Administration of hCG promoted nerve regeneration in vivo and neurite outgrowth and survival of primary neurons in vitro. So far, the function of hCG and its receptor in the nervous system remains unclear.
To understand the role of hCG and its receptor in the development of the mammalian nervous system, we used the bipotent cell line PC12, derived from a rat adrenal pheochromocytoma, as an in vitro model. To examine the effect of hCG on PC12 differentiation, we transduced wild-type human LH/CG-R into PC12 cells and used the stable transgene-expressing subclones. Activation of transduced LH/CG-R with hCG/LH in PC12 cells or expression of a constitutively activated mutated LH/CG-R in PC12 cells induced distinct morphological and biochemical changes characteristic of neuronal differentiation. The differentiation effect of hCG was ligand dose- and time-dependent. Western blot analysis revealed that both the extracellular signal–regulated kinases (ERKs) and p38 mitogen–activated protein kinase (MAPK) were activated after hCG treatment. Inhibitor studies showed that both the ERK and p38 MAPK signal transduction pathways were indispensable for the differentiation process. In addition, the phospholipase C (PLC) pathway was partly involved in hCG-induced PC12 differentiation. Figure 11.3 depicts the relationship among the different pathways. These findings suggest a potential role for hCG/LH and LH/CG-R in the neurogenesis of the mammalian nervous system.
Figure 11.3
Interacting pathways involved in hCG-induced neuronal differentiation.
We also investigated the possible effect of hCG on initiation of myelination. Myelin protein zero (P0) is the major component of the myelin sheath of the peripheral nervous system and is induced at the initial phase of myelination. When combined with forskolin, hCG dramatically increased P0 mRNA expression in a mouse Schwann cell line transiently expressing LH/CG-R in vitro. We are currently examining P0 protein expression. The results suggest that hCG may promote myelination by inducing P0 expression. Currently under investigation, the mechanism of hCG-induced P0 expression could occur via several transcription factors such as krox-20. Our studies show that hCG/LH and its receptor participate in the development and maintenance of the mammalian nervous system. Thus, hCG could be a potential regeneration drug for future treatment of acute neural injuries or neurodegenerative disorders.
Meng XL, Rennert OM, Chan WY. Human chorionic gonadotropin induces neuronal differentiation of PC12 cells through activation of stably expressed lutropin/choriogonadotropin receptor. Endocrinology 2007;148:5865-73.
Molecular basis of differential activity of LH/CG-R with different mutations of the same amino acid residue
The discovery of LH/CG-R with germline- and somatic-activating mutations in patients with testicular tumor raised a question about the tumorigenic potential of mutated LH/CG-R. Two FMPP patients with the Asp578Gly mutation developed Leydig cell neoplasia while we identified a somatic Asp578His activating mutation in a number of testicular tumor patients. Animal studies have so far failed to establish lines of male or female transgenic founder mice carrying LH/CG-R with the Asp578His mutation, thus suggesting that the germline and somatic mutations are inherently different. Given the poor understanding of the mechanisms underlying the different phenotypic manifestations between receptors with mutations of the same amino acid Asp-578, we hypothesized that the differential effect of LH/CG-R mutations results from the activation of different cellular pathways unique to each mutation.
We observed no significant difference between the signal transduction activities of the Gs/adenylyl cyclase/cAMP/PKA and Gq/PLC/DAG/PKC pathways in the two mutant LH/CG-Rs. To explore further the difference in the biological effects of the two mutations, we established an MA-10 Leydig cell model by stably transfecting with the two mutant human receptors LH/CG-R-Asp578Gly and LH/CG-R-Asp578His. By examining gene expression profiles with cDNA microarray and a systems biology approach, we sought to distinguish the wild type and the two mutants by hierarchical clustering and multidimensional scaling analysis. LH/CG-R with Asp578Gly mutation specifically altered the expression of 54 genes while 49 genes were changed in the presence of LH/CG-R with Asp578His. By comparing both mutants to the wild type, we found that 132 genes were differentially expressed. We identified novel regulatory pathways unique to each mutation, with nine networks in LH/CG-R-Asp578Gly–expressing cells and 12 in LH/CG-R-Asp578His–expressing cells. Further analyses showed c-Myc and c-Src to be the key regulators associated with Asp578Gly and Asp578His mutants, respectively. We used molecular and functional assays to confirm the involvement of these two factors. The results provide a novel explanation for the role of LH/CG-R mutation in testicular tumorigenesis.
Genomic and Epigenomic Studies on Abnormal Cell Growth
Genetic and epigenomic studies on testicular germ cell tumors
Testicular tumor is the leading cancer in men between 20 and 39 years of age, accounting for approximately 20 percent of neoplasms in this age group. Despite recent advances in the diagnosis and treatment of testicular tumor, its causes remain unknown. The most common conditions known to be associated with testicular cancer are cryptorchidism, infertility, and overexposure to pesticides, radiation, and cadmium. Hormonal effects on the development of testicular tumors are also well documented. Epidemiological studies in human and animal clearly suggest a genetic predisposition. Various genetic studies have demonstrated genomic abnormalities in testicular tumors, particularly in testicular germ cell tumors (TGCTs). We are engaged in two projects investigating the genetic and epigenetic mechanisms of testicular germ cell tumor development.
TGCTs have been postulated to originate from CIS (carcinoma in situ) or ITGCNU (intratubular germ cell neoplasia unclassified) precursors. The mechanism(s) by which these premalignant precursors initiate and develop into both seminomas and nonseminomas is unknown. Various genetic studies have demonstrated that amplification of a certain portion of chromosome 12p is consistently observed in the evolving germ cell tumor genome. The testis-specific protein Y–encoded (TSPY) gene is a tandemly repeated gene on the short arm of human Y chromosome. Most of its functional transcriptional units have been mapped within the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Several studies have documented the expression of TSPY in the more common forms of TGCT. To investigate the role of TSPY in the pathogenesis of TGCTs, we profiled the transcripts in CIS/ITGCNU and TGCT samples by using GeneChip microarrays from Affymetrix®. Analysis of microarray data demonstrated a correlation between TSPY expression and upregulation of certain chromosome 12p.13 genes. The results support a role(s) for this Y chromosome gene in the pathogenesis of both gonadoblastoma and TGCTs. Our collaborators Chris Y.F. Lau and Yunmin Li have primarily carried out this project while we were responsible for the expression profiling studies.
Figure 11.4
Hypermethylation of the Homeobox (HOX) locus in a testicular tumor sample revealed by tiling array analysis. The different HOX genes are indicated at the lower part of the figure.
DNA methylation is one of the main epigenetic modifications during development. In cancer cells, its disruption is manifested in the regulatory regions (CpG islands) or histone proteins. Aberrant DNA methylation has been reported in TGCT. The two subtypes of TGCT, namely, seminoma and non-seminoma, share similar regional genomic disruptions. On the other hand, recent studies suggested different epigenotypes: CpG island methylation is virtually absent from seminomas while the methylation level in non-seminomas is similar to that of other solid tumors. Despite the observation of aberrant methylation in TGCT, the global picture of epigenetic alteration in the TGCT genome remains unclear. We focus on the epigenetic alterations in non-seminoma. Using ChIP-on-chip with human tiling microarrays from Affymetrix®, we examined the complete genome methylation profile of several testicular cancer cell lines and non-seminoma tissues from patients. More than 40 percent of the TGCT genome demonstrated genome-wide changes in methylation.
Figure 11.5
Extensive hypomethylation of the PSG family locus in testicular tumor sample revealed by tiling array analysis. Only part of the PSG locus is shown. The different PSG genes present in this locus are indicated.
We selected for confirmation two gene clusters with pronounced differential methylation patterns. The first is the homeobox (HOX) gene cluster with extensive hypermethylation (Figure 11.4); the second is the pregnancy-specific beta-1-glycoprotein (PSG) gene cluster with significant hypomethylation (Figure 11.5). The homeobox gene family is involved not only in segmentation during development but also in cell differentiation. Its epigenetic inactivation has been linked to the development of various human malignancies. PSGs are primarily expressed in placenta. However, we observed overexpression of PSGs in a wide variety of tumors and associated cell lines, particularly in colorectal, lung, and brain tumors. We hypothesize that the overexpression of PSGs in tumors may result from the loss of transcription repression by hypomethylation and that PSGs could be used as a marker for TCGTs. We have collected for in situ validation several testicular tumor tissue samples, including archived frozen or paraffin samples of seminomas, nonseminomas, and carcinoma in situ, as well as normal testis tissue samples. The identification of novel epigenetic markers of TGCT, together with the development of user-friendly and sensitive assays, will improve the detection, treatment, and prognosis of this malignancy.
Li YM, Tabatabai ZL, Lee TL, Hatakeyama S, Ohyama C, Chan WY, Looijenga LHJ, Lau YFC. The Y-encoded TSPY protein: a significant marker potentially plays a role in the pathogenesis of testicular germ cell tumors. Human Pathol 2007;38:1470-81.
Effects of mitochondria on nuclear gene expression
Mitochondrial dysfunction is one of the most notable features of cancer cells. Compared with those of normal cells, the mitochondria of rapidly growing tumor cells are fewer in number and smaller in size and exhibit a variety of altered morphologies. Somatic mutations in mitochondrial DNA are found in 30 to 100 percent of all tumors studied. However, the direct link between the molecular etiology and the alterations in mitochondrial function and morphology has not been demonstrated. The role of the altered mitochondrial genome in tumor growth is also unclear. Recent studies with transmitochondrial cybrids revealed that the presence of cancer cell–derived mitochondria could alter the behavior of the host cell. The basis of this observation is unclear. To understand the role of mitochondria in cell behavior, we used the transmitochondrial cybrids approach to examine the effect of cancer cell–derived mitochondria on nuclear gene expression. Preliminary results indicated distinct differences in nuclear gene expression between cybrids with normal mitochondria and cybrids with cancer cell–derived mitochrondria. We also observed marked differences in nuclear gene expression patterns between cybrids harboring different cancer cell–derived mitochrondria that behave differently with respect to tumorigenicity and ATP synthesis. Experiments are under way to elucidate the cause of the phenotypic difference between these cybrids.
Publications Related to Other Work
Horvath A, Mathyakina L, Vong Q, Baxendale V, Pang ALY, Chan WY, Stratakis C. Serial analysis of gene expression (SAGE) of the human adrenal gland: normal tissue and adrenocortical hyperplasia caused by a germline PRKAR1A mutation. J Clin Endocriol Metab 2006;91:584-96.
Ohta S, Lai EW, Morris JC, Pang ALY, Watanabe M, Yazawa H, Zhang, R, Green JE, Chan WY, Sirajuddin P, Taniguchi S, Powers JF, Tischler AS, Pacak K. Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells. Mol Carcinogenesis 2007 [E-pub ahead of print].
Oram SW, Liu XX, Lee TL, Chan WY, Lau YFC. TSPY potentiates cell proliferation and tumorigenesis by accelerating cell cycle progression in HeLa and NIH3T3 cells. BMC Cancer 2006;6:154.
Vong QP, Li YM, Lau CYF, Dym M, Rennert OM, Chan WY. Structural characterization and expression studies on Dby and its homologs in the mouse. J Androl 2006;27:653-61.
COLLABORATORS
Martine Culty, PhD, Georgetown University, Washington, DC
Martin Dym, PhD, Georgetown University, Washington, DC
Patricia Fechner, MD, Stanford University, Palo Alto, CA
George Jeha, MD, Baylor College of Medicine, Houston, TX
Lefkothea P. Karaviti, MD, Baylor College of Medicine, Houston, TX
Maria Kokkinaki, PhD, Georgetown University, Washington, DC
Chris Y.F. Lau, PhD, University of California San Francisco, San Francisco, CA
Michael Yiu-Kwong Leung, PhD, Rush College of Medicine, Chicago, IL
Yunmin Li, PhD, University of California San Francisco, San Francisco, CA
Yewei Ma, PhD, Baylor College of Medicine, Houston, TX
Phil Mosca, MD, PhD, Southwest Medical Center, Oklahoma City, OK
Alan L.Y. Pang, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Owen M. Rennert, MD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
Yan A. Su, MD, PhD, Loyola University, Chicago, IL
Lee-Jun Wong, PhD, Baylor College of Medicine, Houston, TX
For further information, contact chanwy@mail.nih.gov.
