Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-29a Improves Ovarian Function of Mice with Primary Ovarian Insufficiency by Targeting HMG-Box Transcription Factor/Wnt/ß-Catenin Signaling.

Background: Primary ovarian insufficiency (POI) is a female disease characterized by ovarian function loss under 40 years old. Transplantation of exosomes is an encouraging regenerative medicine method that has the potential for restoring ovarian functions post-POI with high efficiency. Therefore, we investigate the therapeutic efficacy and potential mechanisms of human umbilical cord mesenchymal stem cell- (UCMSC-) derived exosomes on ovarian dysfunction post-POI. Methods: The model of POI was established by intraperitoneal injection with 5 mg/kg cisplatin. The mouse ovarian function was detected by measuring the levels of anti-Mullerian hormone, follicle-stimulating hormone, and estradiol and detecting the morphological changes. For in vitro experiments, the characterization and identification of UCMSCs and UCMSC-derived exosomes were done by observation of morphologies and flow cytometry. To exclude the interference effect of nonspecific precipitation substances, UCMSCs were treated with RNase A or RNase A in combination with Triton X-100. Granulosa cell (GC) identification was performed using immunofluorescence. GC proliferation and viability were assessed using 5-ethynyl-2'-deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8), and GC apoptosis was calculated by flow cytometry. Gene expression and protein levels were evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The binding relationship between miR-29a and HMG-box transcription factor (HBP1) was verified by luciferase reporter assays. Results: In vitro, the human UCMSC-derived exosomes carrying miR-29a upregulation promoted the proliferation of GCs and suppressed their apoptosis. In vivo, miR-29a upregulation reserved the mature follicles and restored the ovarian functions. miR-29a targeted HBP1 and negatively regulated its expression. HBP1 upregulation rescued the miR-29a upregulation-induced inhibition in GC apoptosis and inactivated the Wnt/ß-catenin pathway. Conclusion: The exosomal miR-29a derived from human UCMSCs improves the ovarian function by targeting HBP1 and activating the Wnt/ß-catenin pathway.

HMGB1 in renal ischemic injury.

Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H(2)O(2) (oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.

Cellular methods in cartilage research: primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells.

Work in our laboratory has focused on the in vitro culture of both human articular chondrocytes and human mesenchymal stem cells to understand what controls their ability to synthesise an appropriate cartilage-like extracellular matrix containing a predominantly collagen type II fibrillar network embedded in an aggrecan-rich ECM. This review focuses on the methodologies that we have found to be successful with cartilage and bone marrow sources of human cells and comments on the many factors which may enable improved phenotypic performance once the cells are in a fully chondrogenic environment.

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail.

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.

Transcriptional regulation of scar gene expression in primary astrocytes.

The failure of the adult injured spinal cord to support axonal regeneration is in part attributed to the glial scar. Reactive astrocytes constitute a major cellular component of the glial scar and are heterogeneous with respect to the extracellular matrix proteins that they secrete. Astrocytes may produce antiregenerative molecules such as chondroitin sulphate proteoglycans (CSPGs) or proregenerative molecules such as laminin and fibronectin. While many different CSPGs are expressed after spinal cord injury (SCI) they all rely on the same enzymes, xylosyltransferase-I and -II (XT-I, XT-II) and chondroitin 4-sulfotransferase (C4ST) to add the repulsive chondroitin sulfate side chains to their core proteins. We show that XT-I, XT-II, and C4ST are part of a CSPG biosynthetic gene (CBG) battery. Using primary astrocyte cultures and quantitative PCR we demonstrate that TGFbeta2, PDGF, and IL-6 induce the expression of CBGs, laminin and fibronectin by several-fold. We further show that over-expression of the transcription factor SOX9 also strongly induces the expression of CBGs but does not increase the expression of laminin or fibronectin. Correspondingly, SOX9 knock-down in primary astrocytes causes a decrease in CBG and an increase in laminin and fibronectin mRNA levels. Finally, we show that the in vivo expression profiles of TGFbeta2, PDGF, IL-6, and SOX9 are consistent with their potential roles in differentially regulating CBGs, laminin and fibronectin gene expression in the injured spinal cord. This work suggests that SOX9 levels may be pivotal in determining the balance of pro- and anti-regenerative extracellular matrix molecules produced by astrocytes.

Comparative effects of bone morphogenetic proteins and sox9 overexpression on extracellular matrix metabolism of bovine nucleus pulposus cells.

STUDY DESIGN: An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus cells. OBJECTIVE: To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells. SUMMARY OF BACKGROUND DATA: Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair. METHODS: Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7. RESULTS: Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8. CONCLUSIONS: To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.

RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages.

Abstract High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi from interleukin (IL)-1 receptor type I-/-, Toll-like receptor 2 (TLR2-/-) and RAGE-/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mphi, with RAGE as the major activation-inducing receptor.

Induction of IL-10 in rat peritoneal macrophages and dendritic cells by glatiramer acetate.

Glatiramer acetate (GLAT) is a mixture of basic polypeptides that have been shown to suppress experimental autoimmune encephalomyelitis (EAE). As Copaxone, GLAT is approved for the treatment of relapsing-remitting multiple sclerosis (MS). Different immunomechanisms have been suggested to contribute to the beneficial effects of GLAT which rely on blockade of MHC class II molecules or cross-recognition with myelin basic protein (MBP). Because GLAT could also inhibit experimental autoimmunity not related to myelin proteins, we searched for additional, less-restricted immunomodulatory actions of GLAT. Using freshly isolated resident peritoneal macrophages from naive Lewis rats, it is shown that GLAT profoundly modulates cytokine secretion of the cells. In unseparated macrophages (MPhi) and MPhi of low density, GLAT enhanced constitutive and LPS-induced production of interleukin 10 (IL-10) while LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was dose-dependently suppressed by GLAT. Although both basic proteins GLAT and MBP facilitated adherence of MPhi, MBP had opposite effects on cytokine production suggesting unique properties of GLAT. In contrast to MPhi, peritoneal mast cells produced only little amounts of cytokines. The inductive effect of GLAT on IL-10 production by antigen-presenting cells was also observed in bone marrow-derived rat dendritic cells (DCs) which, unlike MPhi, were not suppressed in their production of TNF-alpha. Induction of IL-10 in different antigen-presenting cells is a new immunomodulatory mechanism of GLAT. In part, it goes along with the inhibition of TNF-alpha and may be a common basis for the known beneficial effects of GLAT on various cellular autoimmune responses including MS.

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

Morphine treatment affects the regulation of high mobility group I-type chromosomal phosphoproteins in C6 glioma cells.

High mobility group (HMG) I-type chromosomal phosphoproteins HMG I/Y and HMG I-C were investigated following morphine treatment of C6BU-1 glioma. Cells were labelled with [32P]-orthophosphoric acid. Electrophoretic profiles and autoradiograms of the control cells revealed the presence of HMG I and HMG I-C proteins. HMG Y was not detected. Northern blot analysis showed a single HMG I/Y transcript. Treatment with morphine lowered the [32P]-incorporation in HMG I and HMGI-C proteins and the level of the HMG I/Y transcript. However, it did not change the protein ratios on the Coomassie stained gels. These results suggest that morphine may trigger independent reaction pathways affecting either transcription regulation and/or postsynthetic phosphorylation of the preexisting HMG I-type proteins. In addition, opposing changes in the postsynthetic phosphorylation of HMG 14 and histones H1AB were also noticed.

High mobility group-like protein in bovine milk stimulates the proliferation of osteoblastic MC3T3-E1 cells.

The active component in bovine milk on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified. Growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The molecular weight of the purified protein was 10 kDa. The amino-terminal sequence of this 10-kDa protein was identical to bovine high mobility group protein (HMG) 1. This 10-kDa protein is suggested to be a basic protein and to have an HMG box, a consensus sequence motif among the HMG family. From these results, we named this protein HMG-like protein. HMG is a ubiquitous nonhistone component of chromatin and considered to be implicated in DNA replication. We found this protein in milk, and it showed a growth-promoting activity. We propose the possibility that HMG-like protein existed in milk and plays an important role for neonate in bone formation by activating osteoblasts.

Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

Enhancement of transcription factor, USF, binding to the adenovirus major late promoter: effect of dithiothreitol and high mobility group protein-1.

Up-stream stimulatory factor (USF)1 is a human transcription factor which binds specifically to the E-box in the Ad MLP located at - 58 from the start site. The nature of USF binding on a Ad MLP DNA fragment was investigated in the presence of DTT and also in the presence of purified HMG-1 using electrophoretic mobility shift assay. We show that the binding capacity of USF for the E-box increases significantly with increasing DTT concentrations. At the higher DTT levels, a second USF-DNA complex is formed in which there is co-occupation of both the E-box and the initiator sequence. The stability of the second complex is largely refractory to an excess of unlabeled oligonucleotide which contains the initiator sequence. These findings indicate a cooperative binding interaction between USF ligands bound simultaneously at the E-box and the Inr sequence. Two models are proposed which are consistent with these data. Furthermore, experiments indicate that the presence of HMG-1, a nuclear protein known to influence transcriptional activity, increases USF binding activity at the E-box by as much as 100%. These findings indicate that both reducing conditions and HMG-1 may act as modulators of USF-regulated transcription.

Human testis-determining factor SRY binds to the major DNA adduct of cisplatin and a putative target sequence with comparable affinities.

cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a widely used anticancer drug that is most effective against tumors of the testis. Although cisplatin is believed to mediate its cytotoxicity through the formation of DNA adducts, the precise biochemical mechanisms underlying its antitumor activity and selectivity for testicular tumors remain elusive. Of significance are the high-mobility group (HMG) domain and other proteins that bind specifically to cisplatin-DNA adducts. The present study focuses on the testis-specific HMG domain protein human SRY (hSRY). The full-length hSRY protein and its HMG domain region alone were expressed in Escherichia coli and purified to homogeneity. The affinities and specificities of full-length hSRY and the hSRY-HMG domain for 20 bp DNAs containing a single cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] intrastrand cross-link or a putative hSRY target site in the CD3epsilon gene enhancer (AACAAAG) were determined in electrophoretic mobility shift assays. Full-length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a Kd(app) of 120 +/- 10 nM and exhibited a 20-fold specificity over unmodified DNA. The HMG domain of hSRY was sufficient for this interaction. The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand cross-link with higher affinity [Kd(app) = 4 +/- 0.7 nM] but with lower specificity (5-fold) than the full-length protein. The affinities of full-length hSRY and the hSRY-HMG domain for a single cisplatin-DNA adduct were comparable to those for the putative target sequence AACAAAG. These data suggest that cisplatin-DNA adducts may compete with specific DNA sequences in vivo for the binding of human SRY. A possible role for this testis-specific protein in the cytotoxicity and organotropic specificity of cisplatin for testicular tumors is proposed.

The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome.

A functional interferon-beta gene enhanceosome was assembled in vitro using the purified recombinant transcriptional activator proteins ATF2/c-JUN, IRF1, and p50/p65 of NF-kappa B. Maximal levels of transcriptional synergy between these activators required the specific interactions with the architectural protein HMG I(Y) and the correct helical phasing of the binding sites of these proteins on the DNA helix. Analyses of the in vitro assembled enhanceosome revealed that the transcriptional synergy is due, at least in part, to the cooperative assembly and stability of the complex. Reconstitution experiments showed that the formation of a stable enhanceosome-dependent preinitiation complex require cooperative interactions between the enhanceosome; the general transcription factors TFID, TFIIA, and TFIIB; and the cofactor USA. These studies provide a direct biochemical demonstration of the importance of the structure and function of natural multicomponent transcriptional enhancer complexes in gene regulation.

High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA.

We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.

A 361 base pair region of the rat FSH-beta promoter contains multiple progesterone receptor-binding sequences and confers progesterone responsiveness.

The rat is frequently used as a model to study the role of progesterone (P) in regulating FSH secretion and synthesis. The ability of P to modulate rat FSH-beta mRNA levels suggests the presence of a functional hormone response element. We have found three PRE-like sequences upstream of the transcription start site in the rat FSH-beta gene. These sequences are herein referred to as PRE-like sequence #1, #2 and #3 with #1 being most distal from the start site. The current studies determined whether these PRE-like sequences bound P receptor (PR) and were functional in regulating the induction of expression by P. Electrophoretic mobility shift assays (EMSA) demonstrated that a single 289 base pair (bp) DNA fragment encompassing all three PRE-like sequences specifically bound PR. Further, PR bound with high affinity to double-stranded oligonucleotides representing individual PRE-like sequences #1, #2 and, with lower affinity to a double-stranded oligonucleotide representing PRE-like sequence, #3. We have cloned a 361 bp sequence from the promoter region of the rat FSH-beta gene encompassing all three PRE-like sequences into a luciferase reporter vector (pGL3-promoter) yielding pFSHbeta361-luc+ which when transiently transfected into primary rat pituitary cell cultures, conferred P-responsiveness to a heterologous promoter. P-responsiveness was dependent upon the presence of PR and was blocked by the PR antagonist RU-486. These data strongly suggest the presence of functional PRE's in the rat FSH-beta gene promoter.

Repair of cisplatin--DNA adducts by the mammalian excision nuclease.

Nucleotide excision repair is one of the many cellular defense mechanisms against the toxic effects of cisplatin. An in vitro excision repair assay employing mammalian cell-free extracts was used to determine that the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA adduct, is excised by the excinuclease from a site-specifically modified oligonucleotide 156 base pairs in length. Repair of the minor interstrand d(G)/d(G) cross-link was not detected by using this system. Proteins containing the high mobility group (HMG) domain DNA-binding motif, in particular, rat HMG1 and a murine testis-specific HMG-domain protein, specifically inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occurred in a reconstituted system containing highly purified repair factors. These results indicate that HMG-domain proteins can block excision repair of the major cisplatin-DNA adducts and suggest that such an activity could contribute to the unique sensitivity of certain tumors to the drug. The reconstituted excinuclease was more efficient at excising the 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments using whole cell extracts [Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394-10398]. This result suggests that structural differences among the platinated DNA substrates, and not the presence of unidentified cellular factors, determine the relative excision repair rates of cisplatin-DNA intrastrand cross-links in the whole cell extracts.

Concerted integration of linear retroviral DNA by the avian sarcoma virus integrase in vitro: dependence on both long terminal repeat termini.

We have reconstituted integration reactions in vitro with specially designed donor DNAs, a supercoiled plasmid acceptor, purified bacterium-derived Rous sarcoma virus integrase (IN), and a host cell DNA-bending protein, HMG1. The duplex donor DNAs are approximately 300 deoxynucleotides in length and contain only 15 bp of the RSV U3 and U5 termini at the respective ends. The donor has blunt U3 and U5 termini which end with the sequence 5'CATT. Joining of the donor DNA to the acceptor DNA is detected by using a simple biochemical assay. Integration was found to be dependent on both U3 and U5 termini; mutations in either result in a significant decrease in the level of integration in vitro. Restriction digestion of the products is consistent with most integrants representing a concerted integration in which both long terminal repeat termini come from the same donor molecule. The U5 and U3 sequences in the substrate flank a supF tRNA gene, permitting biological selection of integrants. Many integrants have been sequenced, and have all of the hallmarks of authentic viral integration, including the removal of a terminal TT dinucleotide from each donor DNA end, and duplication of acceptor sequences at the integration site without introducing deletions into the acceptor. Target site selection in the acceptor plasmid was random except that the orientation of integrants selected was apparently influenced by supF transcription. Mutations which substituted the conserved CA dinucleotide with a GA pair led to a decreased rate of integration. In 2 of 14 mutant integrants sequenced, deoxynucleotides were deleted from either the U5 or U3 terminus. In one instance, an internal CA dinucleotide was used, which resulted in a 10-bp U5 donor deletion. In the other, an internal GA dinucleotide was used, which produced a 5-bp U3 donor deletion. Both of these integrants provide further evidence that concerted integration in this reconstituted system requires interactions between IN and the U3 and U5 termini from the same donor molecule.

HMGI(Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.

Expression of the chemokine MGSA/GRO is upregulated as melanocytes progress to melanoma cells. We demonstrate that constitutive and cytokine induced MGSA/GRO alpha expression requires multiple DNA regulatory regions between positions -143 to -62. We have previously shown that the NF-kappa B element at -83 to -65 is essential for basal and cytokine induced MGSA/GRO alpha promoter activity in the Hs294T melanoma and normal retinal pigment epithelial (RPE) cells, respectively. Here, we have determined that the Sp1 binding element located approximately 42 base pairs upstream from the NF-kappa B element binds Sp1 and Sp3 constitutively and this element is necessary for basal MGSA/GRO alpha promoter activity. We demonstrate that the high mobility group proteins HMGI(Y) recognize the AT-rich motif nested within the NF-kappa B element in the MGSA/GRO alpha promoter. Loss of either NF-kappa B or HMGI(Y) complex binding by selected point mutations in the NF-kappa B element results in decreased basal and cytokine induced MGSA/GRO alpha promoter activity. Thus, these results indicate that transcriptional regulation of the chemokine MGSA/GRO alpha requires at least three transcription factors: Sp1, NF-kappa B and HMGI(Y).