Frontline Science: Targeted expression of a dominant-negative high mobility group A1 transgene improves outcome in sepsis.

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis.

Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells.

It has been almost a decade since the last review appeared comparing and contrasting the influences that the different families of High Mobility Group proteins (HMGA, HMGB and HMGN) have on the various DNA repair pathways in mammalian cells. During that time considerable progress has been made in our understanding of how these non-histone proteins modulate the efficiency of DNA repair by all of the major cellular pathways: nucleotide excision repair, base excision repair, double-stand break repair and mismatch repair. Although there are often similar and over-lapping biological activities shared by all HMG proteins, members of each of the different families appear to have a somewhat 'individualistic' impact on various DNA repair pathways. This review will focus on what is currently known about the roles that different HMG proteins play in DNA repair processes and discuss possible future research areas in this rapidly evolving field.

Characterizing metabolic changes in human colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer death worldwide, despite the fact that it is a curable disease when diagnosed early. The development of new screening methods to aid in early diagnosis or identify precursor lesions at risk for progressing to CRC will be vital to improving the survival rate of individuals predisposed to CRC. Metabolomics is an advancing area that has recently seen numerous applications to the field of cancer research. Altered metabolism has been studied for many years as a means to understand and characterize cancer. However, further work is required to establish standard procedures and improve our ability to identify distinct metabolomic profiles that can be used to diagnose CRC or predict disease progression. The present study demonstrates the use of direct infusion traveling wave ion mobility mass spectrometry to distinguish metabolic profiles from CRC samples and matched non-neoplastic epithelium as well as metastatic and primary tumors at different stages of disease (T1-T4). By directly infusing our samples, the analysis time was reduced significantly, thus increasing the speed and efficiency of this method compared to traditional metabolomics platforms. Partial least squares discriminant analysis was used to visualize differences between the metabolic profiles of sample types and to identify the specific m/z features that led to this differentiation. Identification of the distinct m/z features was made using the human metabolome database. We discovered alterations in fatty acid biosynthesis and oxidative, glycolytic, and polyamine pathways that distinguish tumors from non-malignant colonic epithelium as well as various stages of CRC. Although further studies are needed, our results indicate that colonic epithelial cells undergo metabolic reprogramming during their evolution to CRC, and the distinct metabolites could serve as diagnostic tools or potential targets in therapy or primary prevention. Graphical Abstract Colon tissue biopsy samples were collected from patients after which metabolites were extracted via sonication. Two-dimensional data were collected via IMS in tandem with MS (IMMS). Data were then interpreted statistically via PLS-DA. Scores plots provided a visualization of statistical separation and groupings of sample types. Loading plots allowed identification of influential ion features. Lists of these features were exported and analyzed for specific differences. Direct comparisons of the ion features led to the identification and comparative analyses of candidate biomarkers. These differences were then expressed visually in charts and tables.

HMGA1 drives metabolic reprogramming of intestinal epithelium during hyperproliferation, polyposis, and colorectal carcinogenesis.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

Metabolomics of colorectal cancer: past and current analytical platforms.

Metabolomics is coming of age as an important area of investigation which may help reveal answers to questions left unanswered or only partially understood from proteomic or genomic approaches. Increased knowledge of the relationship of genes and proteins to smaller biomolecules (metabolites) will advance our ability to diagnose, treat, and perhaps prevent cancer and other diseases that have eluded scientists for generations. Colorectal tumors are the second leading cause of cancer mortality in the USA, and the incidence is rising. Many patients present late, after the onset of symptoms, when the tumor has spread from the primary site. Once metastases have occurred, the prognosis is significantly worse. Understanding alterations in metabolic profiles that occur with tumor onset and progression could lead to better diagnostic tests as well as uncover new approaches to treat or even prevent colorectal cancer (CRC). In this review, we explore the various analytical technologies that have been applied in CRC metabolomics research and summarize all metabolites measured in CRC and integrate them into metabolic pathways. Early studies with nuclear magnetic resonance and gas-chromatographic mass spectrometry suggest that tumor cells are characterized by aerobic glycolysis, increased purine metabolism for DNA synthesis, and protein synthesis. Liquid chromatography, capillary electrophoresis, and ion mobility, each coupled with mass spectrometry, promise to advance the field and provide new insight into metabolic pathways used by cancer cells. Studies with improved technology are needed to identify better biomarkers and targets for treatment or prevention of CRC.

Why target PIM1 for cancer diagnosis and treatment?

The highly conserved proto-oncogenic protein PIM1 is an unusual serine or threonine kinase, in part because it is constitutively active. Overexpression of PIM1 experimentally leads to tumor formation in mice, while complete knockout of the protein has no observable phenotype. It appears to contribute to cancer development in three major ways when it is overexpressed; by inhibiting apoptosis, by promoting cell proliferation and by promoting genomic instability. Expression in normal tissues is nearly undetectable. However, in hematopoietic malignancies and in a variety of solid tumors, increased PIM1 expression has been shown to correlate with the stage of disease. This characteristic suggests it can serve as a useful biomarker for cancer diagnosis and prognosis. Several specific and potent inhibitors of PIM1’s kinase activity have also been shown to induce apoptotic death of cancer cells, to sensitize cancer cells to chemotherapy and to synergize with other anti-tumor agents, thus making it an attractive therapeutic target.

Distamycin A inhibits HMGA1-binding to the P-selectin promoter and attenuates lung and liver inflammation during murine endotoxemia.

BACKGROUND: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes") that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. OBJECTIVES: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. METHODOLOGY/PRINCIPAL FINDINGS: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. CONCLUSIONS/SIGNIFICANCE: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

Pim-2 phosphorylation of p21(Cip1/WAF1) enhances its stability and inhibits cell proliferation in HCT116 cells.

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21(Cip1/WAF1) (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.

Nuclear functions of the HMG proteins.

Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed.

HMGA1 levels influence mitochondrial function and mitochondrial DNA repair efficiency.

HMGA chromatin proteins, a family of gene regulatory factors found at only low concentrations in normal cells, are almost universally overexpressed in cancer cells. HMGA proteins are located in the nuclei of normal cells except during the late S/G(2) phases of the cell cycle, when HMGA1, one of the members of the family, reversibly migrates to the mitochondria, where it binds to mitochondrial DNA (mtDNA). In many cancer cells, this controlled shuttling is lost and HMGA1 is found in mitochondria throughout the cell cycle. To investigate the effects of HMGA1 on mitochondria, we employed a genetically engineered line of human MCF-7 cells in which the levels of transgenic HMGA1 protein could be reversibly controlled. "Turn-ON" and "turn-OFF" time course experiments were performed with these cells to either increase or decrease intracellular HMGA1 levels, and various mitochondrial changes were monitored. Results demonstrated that changes in both mtDNA levels and mitochondrial mass inversely paralleled changes in HMGA1 concentrations, strongly implicating HMGA1 in the regulation of these parameters. Additionally, the level of cellular reactive oxygen species (ROS) increased and the efficiency of repair of oxidatively damaged mtDNA decreased as consequences of elevated HMGA1 expression. Increased ROS levels and reduced repair efficiency in HMGA1-overexpressing cells likely contribute to the increased occurrence of mutations in mtDNA frequently observed in cancer cells.

HMGA1a: sequence-specific RNA-binding factor causing sporadic Alzheimer's disease-linked exon skipping of presenilin-2 pre-mRNA.

Aberrant exon 5 skipping of presenilin-2 (PS2) pre-mRNA produces a deleterious protein isoform PS2V, which is almost exclusively observed in the brains of sporadic Alzheimer's disease patients. PS2V over-expression in vivo enhances susceptibility to various endoplasmic reticulum (ER) stresses and increases production of amyloid-beta peptides. We previously purified and identified high mobility group A protein 1a (HMGA1a) as a trans-acting factor responsible for aberrant exon 5 skipping. Using heterologous pre-mRNAs, here we demonstrate that a specific HMGA1a-binding sequence in exon 5 adjacent to the 5' splice site is necessary for HMGA1a to inactivate the 5' splice site. An aberrant HMGA1a-U1 snRNP complex was detected on the HMGA1a-binding site adjacent to the 5' splice site during the early splicing reaction. A competitor 2'-O-methyl RNA (2'-O-Me RNA) consisting of the HMGA1a-binding sequence markedly repressed exon 5 skipping of PS2 pre-mRNA in vitro and in vivo. Finally, HMGA1a-induced cell death under ER stress was prevented by transfection of the competitor 2'-O-Me RNA. These results provide insights into the molecular basis for PS2V-associated neurodegenerative diseases that are initiated by specific RNA binding of HMGA1a.

High-mobility group A1 proteins inhibit expression of nucleotide excision repair factor xeroderma pigmentosum group A.

Cells that overexpress high-mobility group A1 (HMGA1) proteins exhibit deficient nucleotide excision repair (NER) after exposure to DNA-damaging agents, a condition ameliorated by artificially lowering intracellular levels of these nonhistone proteins. One possible mechanism for this NER inhibition is down-regulation of proteins involved in NER, such as xeroderma pigmentosum complimentation group A (XPA). Microarray and reverse transcription-PCR data indicate a 2.6-fold decrease in intracellular XPA mRNA in transgenic MCF-7 cells overexpressing HMGA1 proteins compared with non-HMGA1-expressing cells. XPA protein levels are also approximately 3-fold lower in HMGA1-expressing MCF-7 cells. Moreover, whereas a >2-fold induction of XPA proteins is observed in normal MCF-7 cells 30 min after UV exposure, no apparent induction of XPA protein is observed in MCF-7 cells expressing HMGA1. Mechanistically, we present both chromatin immunoprecipitation and promoter site-specific mutagenesis evidence linking HMGA1 to repression of XPA transcription via binding to a negative regulatory element in the endogenous XPA gene promoter. Phenotypically, HMGA1-expressing cells exhibit compromised removal of cyclobutane pyrimidine dimer lesions, a characteristic of cells that express low levels of XPA. Importantly, we show that restoring expression of wild-type XPA in HMGA1-expressing cells rescues UV resistance comparable with that of normal MCF-7 cells. Together, these data provide strong experimental evidence that HMGA1 proteins are involved in inhibiting XPA expression, resulting in increased UV sensitivity in cells that overexpress these proteins. Because HMGA1 proteins are overexpressed in most naturally occurring cancers, with increasing cellular concentrations correlating with increasing metastatic potential and poor patient prognosis, the current findings provide new insights into previously unsuspected mechanisms contributing to tumor progression.

Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins.

Previous work has established that stably transfected human MCF7 cells over-expressing high mobility group A1 proteins (HMGA1) are deficient in global genomic repair (GGR) following exposure to either UV light or cisplatin. To investigate whether HMGA1 over-expression also interferes with gene-specific repair, we employed a rapid and convenient quantitative polymerase chain reaction assay for measuring repair in unique DNA sequences. Efficiency of UV-induced lesion removal was assessed for two genes in MCF7 cells either induced, or not, to over-express transgenic HMGA1 proteins: the constitutively active HPRT gene and the transcriptionally silent beta-globin gene. As controls, similar experiments were also performed in non-transgenic MCF7 cells that do not express detectable levels of HMGA1 and in normal human embryonic fibroblasts that naturally over-express HMGA1 proteins. Our results indicate that exposure of cells to a UV dose of 20 J/m2 produced an average of 0.21+/-0.03 and 0.19+/-0.02 lesions/kb in the HPRT and beta-globin genes, respectively, with no significant difference between HMGA1 over-expressing cells and non-expressing cells. On the other hand, analysis of repair following UV exposure revealed that, compared to controls, HMGA1 over-expressing cells take considerably longer to repair photo-lesions in both the active HPRT and the silent beta-globin loci, with non-expressing cells repairing 50% of lesions in HPRT 3-4 h faster than HMGA1 over-expressing cells. Interestingly, the delay in repair is even more prolonged in the silent beta-globin locus in HMGA1 over-expressing cells compared to control cells. To our knowledge, this is the first report of HMGA1 proteins inhibiting nucleotide excision repair (NER) within specific genes located in either transcriptionally active "open", or inactive "closed", chromatin domains. Furthermore, taken together with previous findings, these results suggest that HMGA1 over-expression interferes with repair processes common to both the GGR and transcription-coupled repair pathways.

The high-mobility group A1 gene up-regulates cyclooxygenase 2 expression in uterine tumorigenesis.

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers.

Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function.

We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential (Delta Psi(m)). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a.

The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation.

Inhibition of nucleotide excision repair by high mobility group protein HMGA1.

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.