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Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia and a propensity for transformation to acute myeloid leukemia (AML). MDS is frequently associated with epigenetic gene silencing via methylation of cytosine residues in gene regulatory regions, and DNA methyl-transferase 1 (DNMT1) inhibitors, such as 5'azacytidine and 5-aza-2'-deoxycytidine (decitabine, DAC), are two of the three agents that are FDA approved for treatment of MDS. Although these drugs are not curative, they induce hematological improvement or improved survival in a significant fraction of MDS patients. Two novel, thiol-substituted 2'-deoxycytidine (dCyd) analogs designated T-dCyd (4'-thio-2'-deoxycytidine) and Aza-T-dCyd (5-aza-4'-thio-2'-deoxycytidine) were synthesized and shown to be potent DNMT1 inhibitors in vitro. We evaluated these drugs in vivo using the NUP98-HOXD13 (NHD13) mouse model for MDS.

Core binding factor (CBF) is a heterodimeric transcription factor comprised of RUNX proteins (RUNX1, RUNX2, RUNX3) and CBF beta. This transcription factor has been shown to play an essential role in the development of various tissues by controlling cell-specific proliferation and differentiation. Our previous studies demonstrated a dramatic upregulation of RUNX1 expression by the LH/FSH surge in periovulatory follicles of rodent ovaries, suggesting a role for this protein in regulating the expression of several genes in the ovulatory cascade in preovulatory granulosa cells. However, little is known about the expression of other components of CBF in the rat ovary. In the present study, we sought to determine 1) the expression pattern of Runx2 mRNA and protein, 2) the DNA binding activity of RUNX2, and 3) the expression pattern of CBF beta using ovaries from eCG/hCG-stimulated immature rats (0, 6, 12 and 24 h post-hCG) as well as cycling rats (during the periovulatory period). First, data from Northern blot analyses and Realtime PCR showed that the level of Runx2 mRNA was increased at 12 and 24 h after hCG injection. In situ hybridization analyses revealed high levels of expression of Runx2 mRNA in periovulatory follicles, newly forming corpora lutea (CL) and CL from previous estrous cycles. Consistent with the expression pattern of Runx2 mRNA, Western blot analyses showed that RUNX2 protein was detected at 12 h after hCG injection and further increased at 24 h post-hCG. The in vitro expression pattern of Runx2 mRNA and protein was also determined in granulosa cells of preovulatory ovaries obtained at 48 h post-hCG and cultured in the absence or presence of hCG at various time points. hCG stimulated the expression of Runx2 mRNA and protein in granulose cell cultures. Second, to determine the transcriptional activity of RUNX2, nuclear extracts isolated from periovulatory ovaries collected at 0, 6, 12, or 24 h after hCG injection were analyzed using a TransAM kit. The binding activity of RUNX2 to a consensus sequence was increased at 24 h after hCG injection. Next, the expression pattern of mRNA for CBF beta, heterodimeric binding partner of RUNX, was analyzed by Northern blot and Realtime PCR using ovaries from eCG/hCG-stimulated immature rats. The CBF beta mRNA expression was readily detected throughout the periovulatory period, yet no change in the level of expression was detected at any of the time points of tissue collection. This expression pattern of CBF beta mRNA was mimicked by the level of CBF beta protein in whole cell extracts from periovulatory ovaries. However, CBF beta protein in nuclear extracts began to increase at 6 h and further increased at 12 h post-hCG, suggesting translocation of CBF beta from the cytoplasm to the nucleus after hCG stimulation. In conclusion, the data from the present study demonstrate that the LH/FSH surge induces Runx2 mRNA and protein expression in periovulatory follicles and CL, consequently increasing the transcriptional activity of RUNX2, while CBF beta is constitutively expressed. In addition, the increase in nuclear expression of CBF beta after hCG stimulation provided evidence that the LH-induced RUNX proteins (both RUNX1 and RUNX2) bind to their partner, CBF beta and move to the nucleus to exert their transcriptional action in the ovary. (supported by NIH NCRR P20 15592 and RO3 HD051727) (poster)

RGC32 was first detected in oligodendrocytes in response to complement activation and has since been implicated as a cell cycle regulator. Recently, we identified Rgc32 as one of several genes in which expression was decreased by knockdown of LH-induced RUNX1 expression via siRNA in cultured rat granulosa cells. We further demonstrated the up-regulation of Rgc32 mRNA expression in periovulatory ovaries from gonadotropin-primed immature rats. These results suggested that Rgc32 might be a possible transcriptional target of RUNX1. In addition, we documented the localization of Rgc32 mRNA in granulosa cells of periovulatory follicles, newly forming corpora lutea (CL) and CL from previous estrous cycles in periovulatory ovaries obtained from naturally cycling adult rats. This finding implied that Rgc32 mRNA expression may be regulated during the luteal period. Therefore, in the present study, we pursued two distinct objectives: 1) to determine whether RUNX1 directly interacts with the Rgc32 gene in periovulatory granulosa cells, and 2) to determine the expression pattern of Rgc32 mRNA during the luteal period in the rat ovary. To investigate the possibility of RUNX1 binding to the Rgc32 gene, a computational analysis using TFSEARCH was performed and revealed multiple RUNX binding sites in the putative promoter region of the rat Rgc32 gene. To verify the interaction of RUNX1 and the Rgc32 gene in vivo, granulosa cells were isolated from periovulatory ovaries (11 h post-hCG) of gonadotropin-primed immature rats and used for ChIP assays. The PCR data revealed the enrichment of a DNA fragment containing RUNX binding sites, providing experimental evidence of direct binding of RUNX1 in the Rgc32 promoter region. Next, to examine the expression of Rgc32 mRNA during the luteal period, immature rats were injected with eCG and 48 h later, hCG to induce ovulation and subsequent pseudopregnancy. Ovaries were collected on 4, 8, 12, 18, 21, or 23 days after hCG injection. Northern blot analysis demonstrated the high expression of Rgc32 mRNA throughout gonadotropin- induced pseudopregnancy and a transient increase in the level of Rgc32 mRNA was observed at 12 days after hCG administration (P < 0.05), a time associated with a decline of luteal progesterone production. To further determine whether this transient up-regulation of Rgc32 mRNA was related with a rapid decline of progesterone production by CL (functional regression), we employed the rat induced-luteal regression model. Gonadotropin-primed rats were injected with bromocriptine at 7 days after hCG administration to induce functional regression and at 10 days after hCG administration with prolactin to induce structural regression of CL. Northern blot analyses demonstrated that levels of Rgc32 mRNA were transiently increased in the ovaries exposed to bromocriptine (P < 0.05). In conclusion, findings of the present study provided evidence of transcriptional regulation of the Rgc32 gene by RUNX1 in periovulatory granulosa cells. Results from the present study also demonstrated that Rgc32 mRNA level is transiently increased during functional regression of CL. The functional importance of RGC32 expression in the ovary has yet to be determined. (supported by NIH NCRR P20 15592 and RO3 HD051727) (poster)

In response to the LH/FSH surge, a preovulatory follicle undergoes dramatic changes that culminate in ovulation and luteal formation. These changes are caused by actions of specific transcription factors that control periovulatory gene expression. Our previous studies showed that Runx1and Runx2expression were induced by the LH surge in periovulatory ovaries. Runx1expression was transient in periovulatory follicles and declined rapidly before ovulation, whereas Runx2expression was increased in periovulatory follicles and newly forming CL. Runx1and Runx2were also highly expressed in periovulatory COCs. In an attempt to identify the downstream targets of RUNX2, we performed microarray analyses using Runx2silenced granulosa cells. Interestingly, the microarray data revealed increased levels of Runx1mRNA in Runx2siRNA-treated cells. Further analyses of the Runx1gene revealed 2 consensus RUNX binding sites in the Runx1promoter region. Taken together with the inverse pattern of expression between Runx2and Runx1during the late periovulatory period, we hypothesized that RUNX2 plays a role in down-regulation of Runx1expression in luteinizing granulosa cells and/or expanding cumulus cells. To test this hypothesis, preovulatory granulosa cells or COCs were collected from eCG-primed immature rat ovaries and cultured with various reagents. First, to confirm the microarray data, granulosa cells were transfected with negative siRNA or Runx2siRNA and stimulated with agonists (forskolin + PMA) for 24 or 48 hr to induce Runx2expression. Real-time PCR and Western blot analyses confirmed the knockdown of Runx2expression and increases in Runx1expression in Runx2siRNA-treated cells. In addition, the levels of mRNA and protein for Ptgs2and Tnfaip6were also increased in Runx2silenced cells. Second, to assess whether Runx2over-expression can inhibitRunx1expression, granulosa cells were infected with adenovirus expressing Runx2(Ad-RUNX2). We found that Runx2over-expression reduced the agonist-stimulated Runx1, Ptgs2, and Tnfaip6expression. Next, to determine whether RUNX2 regulates Runx1expression at the transcriptional level, Runx1promoter constructs were generated and transfected into granulosa cells. Luciferase reporter assays revealed that mutation of RUNX binding sites in the Runx1promoter resulted in further induction of agonist-stimulated promoter activity of the Runx1gene. Lastly, to determine whether RUNX2 regulates Runx1expression in cumulus cells, COCs were stimulated with forskolin + amphiregulin to induce expansion. These agonists induced full expansion of COCs and increased the expression of COC expansion-related genes (Ptgs2, Tnfaip6, Has2, Ptx3and Cspg2) as well as Runx1. When COCs were infected with Ad-RUNX2, the agonist-stimulated expression of Runx1, Ptgs2, Tnfaip6, Has2,and Cspg2, but not Ptx3, was decreased compared to that in control COCs. Moreover, the over-expression of RUNX2 reduced COC expansion. Taken together, these data demonstrated that RUNX2 regulates the expression of Runx1, Ptgs2, Tnfaip6, Has2,and Cspg2in luteinizing granulosa cells and/or expanding cumulus cells. This novel finding suggests that RUNX2 contributes to the down-regulation of Runx1and several COC expansion genes during the periovulatory period to support proper COC expansion and luteinization. (supported by P20 RR15592, R01HD061617 and R03HD066012.)(poster)

The matrix metalloproteinase (MMP) family is comprised of proteolytic enzymes that remodel the extracellular matrix (ECM). These MMP members reflect vast biological roles, including the extensive remodeling associated with follicular rupture. Mmp10, a member of the stromelysin MMP sub-family, has been shown to be highly expressed in numerous cancer types and to correlate with inflammatory responses. However, little is known about the role of Mmp10in the ovary. In the present study, Mmp10expression was analyzed in the periovulatory rat ovary, in vitro cultured rat granulosa cells and human periovulatory granulosa cells. In the periovulatory rat model, follicular stimulation was initiated in immature female rats (21 days old) by pregnant mare serum gonadotropin hormone (PMSG). Forty-eight hours after stimulation by PMSG, rats were then injected with human chorionic gonadotropin (hCG) to induce ovulation. In this experimental model, ovulation occurs at approximately 14 to 16h following hCG administration. Thus whole ovaries were collected at 0, 6, 12 and 24h after hCG to determine changes in Mmp10mRNA throughout the periovulatory period. For rat granulosa cells, ovaries were collected 48h following PMSG injection; granulosa cells were then isolated and cultured in the absence or presence of hCG for 0, 9, and 24h. Ovaries and granulosa cells were frozen for analysis of Mmp10mRNA expression. To analyze Mmp10mRNA expression in human granulosa cells, granulosa cells were first collected from patients undergoing elective surgery at different periovulatory periods (preovulatory, early ovulatory and late ovulatory phase groups). Surgery was performed on the patients at the preovulatory phase, prior to the LH surge, without the administration of hCG. The remaining patients received an injection of 250 ug hCG to mimic the endogenous LH surge and underwent surgery after varying lengths of time following hCG injection: early ovulatory phase 12 to ≤18h and late ovulatory phase >18 to ≤34h. Granulosa cells were frozen and Mmp10mRNA expression analyzed by microarray profiles. For the rat, RNA was isolated from ovaries or granulosa cells and quantitative Real-Time PCR analysis was carried out. Administration of hCG induced the expression of Mmp10in the rat ovary prior to ovulation at 6h post-hCG and expression remained elevated through 24h post-hCG. In cultured granulosa cells, Mmp10expression increased in both control (untreated) and hCG treated cells after 9h of culture before declining to 0h levels after 24h of culture. The observation that Mmp10mRNA increases independent of hCG hormonal stimulation suggests the stress of cell isolation and/or culture induces Mmp10. Based on the induction of Mmp10observed in the rat ovary, a microarray experiment was conducted to determine the expression in the human. Mmp10expression increased 150-fold between the pre- and late ovulatory phase in human granulosa cells. Thus the increase in Mmp10prior to ovulation in human granulosa cells is similar to findings in the rat ovary indicating an increased expression of Mmp10mRNA after hCG stimulation. In summary, these results suggest that the induction of Mmp10prior to ovulation, in both the rat model and human granulosa cells, may reflect a role of Mmp10in both the late stages of follicular growth as well as the breakdown of the follicular wall during the periovulatory process.(poster)

One of the hallmarks of the LH induced granulosa-luteal transition is a conversion from a proliferative, estrogen producing granulosa cell to a differentiated, progestin secreting luteal cell. The B Cell Translocation Gene (BTG) family has been reported to be involved in cell differentiation, antiproliferation, and gene transcription regulation. We hypothesized that BTGs are involved in this process, however, very little is known about them in the ovary. The present study investigated the spatiotemporal expression pattern, regulation and potential function of BTG1, BTG2, and BTG3 in the rat ovary during the periovulatory period. Immature 22-23 days old rats were injected with eCG (10 IU) s.c. to stimulate follicular development and 48h later with hCG (5 IU) s.c. to induce ovulation and subsequent formation of corpora lutea (CL). Animals were killed at different time points after hCG administration (n=3/time point). Ovaries were collected for isolation of total RNA or protein, or processed for in situ hybridization. Granulosa cells were also collected in vivo at different time points for isolation of total RNA or protein. Quantitative real-time PCR analysis revealed that mRNA for Btg1, Btg2, and Btg3 was highly induced both in intact ovaries and granulosa cells by 4 to 8h after hCG stimulation. The expression of Btg1 mRNA in whole ovary was highest between 4 and 8h before decreasing to control levels by 12 and 24h after hCG. The levels of Btg1 mRNA exhibited a similar expression pattern in the granulosa cells (in vivo) to that observed in the whole ovary with the highest expression at 8h. Btg2 mRNA expression in whole ovaries increased 6.5 fold at 4 and 8h after hCG but did not decrease to control levels by 24h, whereas its expression in the granulosa cells (in vivo) continued to increase until 24h after hCG treatment. Btg3 mRNA levels increased 3 fold at 8h after hCG and remained elevated throughout the periovulatory period in both granulosa cells (in vivo) and the whole ovary. In situ hybridization showed that Btg1 mRNA expression was induced in the theca and granulosa cells of some follicles at 4h after hCG injection. Strikingly, there was a switch in the expression from the theca to the granulosa cell compartment at 8 and 12h after hCG. After ovulation, Btg1 mRNA expression was similar to the 0h ovary with little detectable signal in the forming CL. In contrast, Btg2 mRNA expression level was high in luteal cells at 24h after hCG administration. Btg3 mRNA was induced at 8h in granulosa cells after hCG treatment. Regulation of Btg expression was then examined using cultured granulosa cells. Inhibition of progesterone action and the EGF pathway did not change Btg1 and Btg2 mRNA expression, whereas inhibition of prostaglandin synthesis or RUNX activity diminished Btg2 mRNA levels. To determine the function of the BTGs, overexpression of BTG1 or BTG2 resulted in an increase in the percentage of granulosa cells in the G0/G1 phase, whereas the percentage of cells in the S phase decreased. The apoptosis rate in the granulosa cells overexpressing BTG1 or BTG2 also decreased compared to cells infected with the control GFP vector. In conclusion, the induction of BTG1, BTG2, and BTG3 expression after hCG administration and the ability to arrest the cell cycle may be related to theca and granulosa cells differentiation or the establishment of quiescence. (Supported by NIH RR15592 and AG17164)(poster)

The incidence of iron deficiency anaemia in 6∼24 month old infants due to increase in iron demand for growth spurt is reported ranged 10 to 40%. However this age group has a common acute illness such as urinary tract infection, pneumonia, and other viral infections. The aim of this study is to evaluate that iron parameter and acute phase reactant are useful parameters in differentiating anaemia by infection from anemia by iron deficiency and the mixed anaemia of these. Among 6–24 months of the infants who visited Gyeongsang Univeristy Hospital for 7 years from 2000 to 2006, 131 infants were enrolled. Hemoglobin(Hb), serum ferritin(SF), serum transferrin receptor(STfR), C reactive protein(CRP), interleukin-6(IL-6), prohepcidine were checked. The subgroup of anaemia of inflammation(AI) was defined as Hb <11 g/dL and SF >50 μg/L, the subgroup of iron deficiency anaemia(IDA) as Hb <11 g/dL and SF <12 μg/L and the normal group as Hb ≥11 and SF ≥12 μg/L. The mean STfR in the subgroup of AI, IDA and normal was 3.89(±2.64), 10.6(±4.95) and 3.96(±1.24), respectively. The mean STfR/Log SF of subgroup was 1.87(±1.55), 36.11(±71.5), 2.31(±0.97), respectively. The mean Log(STfR/SF) was statistically significant between 3 subgroup. All IDA group had Log(STfR/SF) >2.55 whereas in all subjects classified as AI it was <2.55, thus clearly separating two. The mean IL-6 of AI was significantly higher than IDA subgroup and the mean prohepcidine of AI was significantly lower than the normal group. Calculating Log(STfR/SF) is a useful criteria in classification of the iron status. Prohepcine has nothing to do with AI. Iron signal predominant over inflammatory signal in AI. The Mean(±SD) of STfR, STfR/LogSF, Log (TfR/SF), CRP, IL-6 and Prohepcidine in Subgroups. AI IDA Normal Same letters mean that are not significantly different (P <0.05) AI, anaemia of inflammation; IDA, iron deficiency anaemia Subgroup(%) 33(25) 29(22) 69(53) Hg(g/dL) <11 <11 ≥11 SF(μg/L) >50 <12 ≥12 STfR mean(±SD) 3.89(2.64)a 10.6(4.95) b 3.96(1.24) a STfR/LogSF mean (±SD) 1.87(1.55) a 36.11(71.5) b 2.31(0.97) a Log (TfR/SF) mean(±SD) 1.30(0.56) a 3.29(0.43) b 1.76(0.43) c CRP mean(±SD) 28(39.2) a 7.6(9.6) b 17(28.0) a IL-6 mean(±SD) 6.1(10.5) a 2.0(6.3) b 4.78(11.3) c Prohepcidine mean(±SD) 204(70.5) a 234(144) a 301(120.6) b Fig.1. Log(TfR/SF) in the subgroup. AI, anaemia of inflammation; IDA, Iron deficiency anaemia. Dotted lines indicate the cut-off value at Log(TfR/SF)=2.55 Fig.1. Log(TfR/SF) in the subgroup. AI, anaemia of inflammation; IDA, Iron deficiency anaemia. Dotted lines indicate the cut-off value at Log(TfR/SF)=2.55

The incidence of iron deficiency anaemia in 6∼24 month old infants due to increase in iron demand for growth spurt is reported ranged 10 to 40%. However this age group has a common acute illness such as urinary tract infection, pneumonia, and other viral infections. The aim of this study is to evaluate that iron parameter and acute phase reactant are useful parameters in differentiating anaemia by infection from anemia by iron deficiency and the mixed anaemia of these. Among 6–24 months of the infants who visited Gyeongsang Univeristy Hospital for 7 years from 2000 to 2006, 131 infants were enrolled. Hemoglobin(Hb), serum ferritin(SF), serum transferrin receptor(STfR), C reactive protein(CRP), interleukin-6(IL-6), prohepcidine were checked. The subgroup of anaemia of inflammation(AI) was defined as Hb <11 g/dL and SF >50 μg/L, the subgroup of iron deficiency anaemia(IDA) as Hb <11 g/dL and SF <12 μg/L and the normal group as Hb ≥11 and SF ≥12 μg/L. The mean STfR in the subgroup of AI, IDA and normal was 3.89(±2.64), 10.6(±4.95) and 3.96(±1.24), respectively. The mean STfR/Log SF of subgroup was 1.87(±1.55), 36.11(±71.5), 2.31(±0.97), respectively. The mean Log(STfR/SF) was statistically significant between 3 subgroup. All IDA group had Log(STfR/SF) >2.55 whereas in all subjects classified as AI it was <2.55, thus clearly separating two. The mean IL-6 of AI was significantly higher than IDA subgroup and the mean prohepcidine of AI was significantly lower than the normal group. Calculating Log(STfR/SF) is a useful criteria in classification of the iron status. Prohepcine has nothing to do with AI. Iron signal predominant over inflammatory signal in AI.

T-cell/histiocyte-rich large B cell lymphoma (THR-BCL) is morphologic variants and subtypes of diffuse large B cell lymphoma (DLBCL). Four immunomorphologic criteria were applied to distinguish THR-BCL from DLBCL. a diffuse or vaguely nodular pattern of neoplastic infiltrate, the presence of scattered large atypical lymphoid cells that are of B-cell phenotype, predominance of a reactive background infiltrate, composed of both T-cell and non-epithelioid histiocytes, minimal presence of small B-cells in neoplastic areas. Bcl-6 protein expression, which was generally accepted markers of germinal center differentiation, was noted more than half of the THR-BCL cases. THR-BCL predominantly affected middle-aged men who present with advanced disease and an unusually high percentage of bone marrow (BM) involvement. Here we report a pediatric case of THR-BCL. 14-year-old boy was presented with both cervical mass which was observed 1 month ago. He complained generalized weakness and chest discomfort had fever. Cervical lymph node and BM biopsy were taken. Laboratory finding was as follows; Hemoglobin 10.4 mg/dL, hematocrit 31 %, leukocyte 6,730/μL, platelet 219,000/μL. Na 128 mmol/L, K 4.6 mmol/L, Cl 95.4 mmol/L, BUN 10 mg/dL, Cr 1.2 mg/dL, LDH 2,108 U/L. Lymph node specimen showed diffuse large B cell lymphoma and numerous histiocytes. Immnuohistochemical staining; CD 20(+), CD 10(−), CD 3(+), CD 68(+). Ann Arbor stage is IVB, international prognostic index is high-intermediate risk. He received induction chemotherapy with prednisolone, vincristine, cyclophosphamide, doxorubicin, rituximab and achieved remission and now he have continued chemotherapy for 7 months in state of complete remission. THR-BCL is a distinct clinicopathologic entity within DLBCL that is characterized by an aggressive behavior and high risk of treatment of failure. That is the reason why delineate this subgroup. Experimental therapeutic strategy was indicated in patients who relapsed after having obtained a complete remission or had partial response after initial chemotherapy.

T-cell/histiocyte-rich large B cell lymphoma (THR-BCL) is morphologic variants and subtypes of diffuse large B cell lymphoma (DLBCL). Four immunomorphologic criteria were applied to distinguish THR-BCL from DLBCL.