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Cancer Prevention Think Tank: Fulfilling the Potential for Disease Control
he broadest measure of cancer prevention research is finding ways to reduce the incidence, progression, recurrence, and metastasis of the disease. This comprehensive, global “reach” of prevention was examined at the first Cancer Prevention Think Tank on December 8, 2006, at NCI-Frederick. Hosted by CCR and organized by Nancy Colburn, PhD, Chief of the Laboratory of Cancer Prevention (LCP), the forum engaged scientists and clinicians from CCR, NCI’s Division of Cancer Prevention (DCP), Baylor College of Medicine, and Brigham and Women’s Hospital for a day-long exchange of information, insights, and strategies. The objective of the Cancer Prevention Think Tank was to identify the most promising, novel molecular targets and interventions to reduce the burden of cancer at every juncture for the general population, high-risk groups, and patients. The meeting was also an opportunity to strengthen collaboration and foster communication among members of the LCP, the Molecular Targets Development Program (MTDP), the Mouse Models of Mammary Cancer Collective (MMMCC), the Animal Models Initiative (AMI), the Inflammation and Cancer Initiative (ICI), and the DCP. In my opening remarks, I highlighted cancer prevention as one of CCR’s core competencies in delivering meaningful advances that benefit at-risk populations and patients. Our efforts to discover molecular targets and pioneer novel interventions effectively combine and leverage expertise from diverse fields to speed progress from elucidation to translation to treatment. The Cancer Prevention Think Tank is an acknowledgement of how important an integrated, multidisciplinary approach is for accelerating our progress toward reducing cancer incidence and mortality. The day began with Powel Brown, MD, PhD, from the Breast Center at Baylor College of Medicine, discussing novel molecular targets for the prevention of breast cancer. Based on his clinical perspective of breast cancer prevention, risk management, and genetics, combined with his research in cancer preventive agents, Dr. Brown stressed the need for combination therapies and effective agents for preventing estrogen receptor (ER)–negative breast cancer. He focused on receptor-selective retinoids, particularly rexinoid bexarotene, for inhibiting growth in breast cancer and premalignant cells; receptor tyrosine kinase inhibitors (TKI); and activator protein-1 (AP-1) transcription factor inhibitors. Dr. Brown’s discussion of validated and potential targets concluded with stem cell inhibitors as an exciting prospect for breast cancer prevention research. Think Tank participants then shared findings on molecular targets for cancer prevention, including targeting translation initiation to prevent turmorigenesis and invasion, the therapeutic potential of programmed cell death (Pdcd) 4 designer drugs, ER zinc finger interventions for the treatment of breast cancer, the role of Lsh and genomic demethylation in tumor progression, and selenoproteins as anticarcinogenic agents. The roundtable discussion that followed focused on targeting the microenvironment for tumor suppression. Angiogenesis, macrophages, and basophils were cited as high-impact targets, in addition to stromal epithelial interactions, epithelial-mesenchymal transition, and myofibroblasts. The consensus was that infection, cytokines, signaling molecules, and transcription factors are critical avenues for studying microenvironment targets. The participants also tackled prospects for targeting cancer stem cells without harming normal stem cells, with Dr. Brown noting the importance of identifying differences between a normal stem cell and a quiescent stem cell undergoing transformation. Insights were also shared about humanizing mice for enhancing physiological responses to drugs. The afternoon guest speaker was Monica Bertagnolli, MD, a surgeon from Brigham and Women’s Hospital, whose clinical practice is integrated with her research to identify markers of colon carcinogenesis and to exploit those targets for tumor prevention and treatment. Dr. Bertagnolli discussed selective cyclooxygenase-2 (COX-2) inhibition for prevention of colorectal adenomas from her standpoint as principal investigator of an NCI-sponsored multi-institutional clinical trial testing this drug. She then described 15-hydroxyprostaglandin dehydrogenase (PGDH) as a new target for chemoprevention and elaborated on prospects for targeting the Indian Hedgehog (IHH) gene and prostaglandin E2 (PGE2) for cancer prevention. The second half of the Think Tank workshop featured presentations on dietary interventions for the prevention of colorectal and other cancers, as well as the inhibition of basic leucine zipper (bZIP) protein transcription factors. Critical points for intervention were identified, including detection of micrometastases and tumor cell dormancy prior to a proliferative phase. During the roundtable discussion, Dr. Bertagnolli stressed the need for “on/off” mechanisms to avert toxicity and drug resistance when a major pathway is chronically suppressed. The participants agreed on the need for more tissue-based studies in people at high risk, with the results subsequently correlated with animal studies. The exchange culminated in a suggestion that CCR basic and clinical researchers team up to study organ-specific cancers, which would complement ongoing investigations focused on transcription factors and signal transduction, for example. The 2006 Cancer Prevention Think Tank highlighted the value of CCR’s efforts toward cancer prevention. The event brought together molecular biologists, mouse geneticists, developmental biologists, epidemiologists, clinical oncologists, and others to prioritize opportunities that have emerged from recent discoveries of molecular events in carcinogenesis, and to guide efforts for exploring new ones. Through initiatives such as the Think Tank, CCR’s research strengths are being leveraged and optimized so that, ultimately, no segment of the population will have unmet needs related to cancer. For more information on CCR’s cancer prevention research, visit LCP’s Web page at http://ccr.cancer.gov/labs/lab.asp?labid=169. Plans are under way to develop an interactive Web site to further stimulate ongoing communication and interaction between Cancer Prevention Think Tank researchers and current and future collaborators.
Cardiac Mitochondrial Compromise in 1-Year-Old Erythrocebus patas Monkeys Perinatally Exposed to Nucleoside Reverse Transcriptase Inhibitors
Clinical manifestations of mitochondrial toxicity include fatigue, lactic acidosis, cardiac and skeletal muscle myopathy, and abnormal results on an echocardiogram. Morphologically damaged mitochondria, mitochondria with altered oxidative phosphorylation (OXPHOS) capacity, and changes in mtDNA quantity have been found in patients with these signs and symptoms. In HIV-1–uninfected infants exposed to NRTIs, clinical evidence of mitochondrial dysfunction is infrequent. However, two HIV-1–uninfected children, born to mothers receiving AZT and 3TC during pregnancy, died at about 1 year of age from severe persistent mitochondrial toxicity, and symptoms of mitochondrial dysfunction have been found in 26 children from a cohort of 2,644 NRTI-exposed children (Barret B et al. AIDS 17: 1769–85, 2003). Furthermore, evidence of mitochondrial morphological damage, revealed by electron microscopy (EM) and mtDNA depletion, has been reported in a large fraction of clinically normal infants born to HIV-1–infected women receiving NRTI therapy during pregnancy (Divi RL et al. AIDS 18: 1013–21, 2004). The mtDNA depletion appears to persist for up to 2 years of age (Poirier MC et al. J Acquir Immune Defic Syndr 33: 175–83, 2003). It is therefore important to evaluate the potential for NRTI-induced mitochondrial toxicity in these children and relevant animal models. We investigated cardiac mitochondrial integrity in 1-year-old Erythrocebus patas monkeys exposed perinatally to human-equivalent NRTI dosing protocols. The patas monkey model has the advantage that its drug pharmacokinetics are similar to those in humans, and drug effects can be examined in the absence of confounding viral infection. To mimic human clinical exposures, the NRTIs were given to both the dam for the last half of gestation and to the newborn for the first 6 weeks of life. Exposures included no drug (n = 4), AZT (n = 4), AZT/3TC (n = 4), AZT/ddI (n = 4), and d4T/3TC (n = 4). The study included assessments of maternal and infant clinical chemistry and cardiac function by echocardiogram before and after parturition. Heart tissue, taken from 1-year-old offspring, was examined for mitochondrial morphology (via EM), mtDNA quantitation, and OXPHOS enzyme-specific activities. Functionally, no difference was observed between unexposed and NRTI-exposed patas infants, as they all behaved similarly and had normal echocardiogram results. In addition, OXPHOS enzyme activities were similar in heart mitochondria from all groups. However, EM studies revealed significant mitochondrial morphological damage in hearts from NRTI-exposed animals compared with unexposed animals (P < .05) (Figure 1). We found mitochondrial proliferation, swollen and disrupted cardiac myofibrils, visibly misaligned sarcomeres, partial or complete erosion of mitochondrial membranes, and replacement of cristae with clear space (Figure 1, parts B and C). Clusters of highly damaged cardiac mitochondria, suggestive of clonal expansions, were observed in the hearts of all NRTI-exposed patas infants but not in the hearts of unexposed controls (Figure 1, part C). In addition to the mitochondrial morphological damage, levels of mtDNA were elevated in the hearts of all NRTI-exposed monkeys, compared with controls (AZT/ddI > AZT/3TC > AZT > d4T/3TC > control; P < .05). Figure 1. Photomicrographs of Erythrocebus patas cardiac tissue at 1 year of age, with no drug exposure (A) and after in utero (10 wk) and post-birth (6 wk) exposures to zidovudine (AZT) plus lamivudine (3TC) (B), and stavudine (d4T) plus 3TC (C). This study showed that, during the first year of life, growing hearts from patas infants exposed perinatally to NRTIs regained the OXPHOS enzyme functionality that was altered in similarly exposed monkeys at birth. However, at 1 year of age, patas monkey hearts showed increases in mtDNA quantity and mitochondrial morphological damage by EM. Mitochondria with altered morphology were clustered in groups suggestive of clonal expansions of mitochondria that had sustained pathologic mutations and/or deletions. Similar damage has been found in AZT-exposed aging mice (Walker DM et al. Cardiovasc Toxicol 4: 133–53, 2004). This study demonstrates that transplacental NRTI exposures in non-human primates results in persistent cardiac pathology on a molecular level. The data suggest that, in the absence of intervention, cardiac insufficiency may arise later in life in NRTI-exposed patas monkeys and humans.
Novel Genetic Test Predicts the Development of Cervical Cancer
Infection with human papillomavirus (HPV) can cause cervical cancer, and more than 70% of early dysplastic lesions carry the virus. For this reason, tests for the detection of HPV genomes have been pursued with the hope of developing a biomarker that allows for the discernment of lesions with low and high risk for disease progression. This goal has been only partially achieved: A test was developed that is very sensitive, in that HPV-negative lesions have a low risk of disease progression. However, only a small fraction of early dysplastic HPV-positive lesions actually progress to severe degrees of dysplasia and cancer. When translated into clinical practice, this means that a positive HPV test does not substantially add to the therapeutic decision-making process. Therefore, tests that can identify the 10% to 15% of the ASCUS (atypical squamous cells of unknown significance) and low-grade dysplastic lesions that have a likelihood of progressing to higher grades of disease would be helpful. A specific marker would not only enable clinicians to identify patients at high risk for progression at an early stage of the disease, it would also assist in tailoring diagnostic and therapeutic procedures for those women with low or no risk of progression. This could reduce the need for more invasive tests, including colposcopy, surgical biopsy, and conization and, consequently, lessen distress for the patient and lower health care costs. The molecular cytogenetic analysis of cervical cancer progression has revealed that the acquisition of extra copies of the long arm of chromosome 3 appears to be a mandatory genetic event: More than 95% of invasive cervical carcinomas carry this specific genomic imbalance (Heselmeyer K et al. Proc Natl Acad Sci U S A 93: 479–84, 1996). The region on chromosome arm 3q most frequently gained contains the gene that encodes the RNA component of human telomerase (TERC), which is intricately involved in cell immortalization and cancer. Based on these results, we hypothesized that the detection of increases in TERC gene copy number in cytological samples might be useful for the genetic diagnosis of cervical dysplasia. We therefore developed and validated a three-color FISH (fluorescent in situ hybridization) probe set that allows enumeration of genomic copy numbers of TERC, along with centromere-specific control probes that recognize chromosomes 3 and 7. Our data showed that the visualization of additional copies of TERC serves as a specific and sensitive test for the diagnosis of cervical dysplasia in routinely collected cytological samples, independent of the morphological assessment (Heselmeyer-Haddad K et al. Am J Pathol 163:1405–16, 2003; see also Frontiers in Science, July 2004, Volume 3, available at http://ccr.cancer.gov/news/frontiers/July_2004.pdf). The goal of the current study was to investigate whether copy number increases of TERC could assist not only in detecting high-grade lesions, but also in assessing progression risk in early, low-grade, dysplastic lesions. We therefore applied the probe cocktail to a series of 59 previously stained archival Pap smears for which repeat smears and clinical follow-up were available. The samples were divided into three groups: (1) cases with cervical intraepithelial neoplasia, grades 1 and 2 (CIN1 and CIN2), that progressed to CIN3; (2) cases with CIN1 and CIN2 that regressed spontaneously; and (3) cases with a normal Pap smear that subsequently showed CIN3 lesions or cervical cancer within a period of only 1 to 3 years. Based on our genetic progression model of cervical cancer, we hypothesized that low-grade lesions that showed progression (group 1) already contained extra copies of TERC, whereas the absence of genomic amplification of this gene would define those early lesions that regressed spontaneously (group 2). In addition, we surmised that at least some of the morphologically normal samples in group 3 contained cells with TERC gains. Indeed the results showed that a third (4 of 12) of the samples in group 3, which were all assessed as cytologically normal, revealed copy number increases of TERC. This demonstrated that phenotypically normal cells can contain TERC amplification, which ultimately leads to the development of invasive disease. In group 1, 7 of the 12 samples showed TERC gains in the initial CIN1/CIN2 lesion; the remaining 5 had a substantial number of cells with four signals for all probes in the panel, including TERC. This signal pattern is consistent with a tetraploidization of the genome, which can be a consequence of HPV infection. In group 2, 7 of the 10 samples that showed regression exhibited normal signal counts (diploidy) for all probes in the panel. Three samples showed a proportion of tetraploid cells (pattern 4-4-4) in 20% to 40% of the cells, with the remaining cells being diploid. None of the lesions that spontaneously regressed showed a gain of TERC. This is consistent with the hypothesis that disease progression does not occur without genomic amplification of TERC. Examples of the results of the FISH test along with the cytological staining pattern are provided in Figure 1. Figure 1. A) Hybridization of the TERC gene (yellow) to previously stained routine Papanicolaou (Pap) smears from a patient who later showed regression to normal cytology. This Pap smear was assessed as Pap IIID (cervical intraepithelial neoplasia [CIN1]). Note that the morphologically suspicious cells do not carry extra copies of the TERC genes (two copies per cell only). Two distinct areas of the slide are presented. B) Hybridization of the TERC gene (yellow) to previously stained routine Pap smears from a patient who showed progression. This sample was assessed as Pap IIID (CIN2). Multiple nuclei that appeared aberrant during the cytological screening throughout the slide reveal extra copies of TERC. Note that both larger nuclei and cells with small nuclei reveal increased copy numbers for this gene (lower right area). C) Hybridization of the TERC gene (yellow) to previously stained routine Pap smears from a patient who eventually experienced disease progression. This patient was initially diagnosed with Pap IIID (CIN1, October 2000), but the patient’s disease was considered to have regressed because subsequent Pap smears were normal (2001). However, in 2002 the follow-up Pap smear was assessed as CIN2, and in 2003 as CIN3. Note multiple 3q-positive cells in the sample. D) Hybridization of the TERC gene (yellow) to previously stained routine Pap smears from another patient who eventually experienced disease progression. The patient’s samples were repeatedly judged as morphologically normal, yet she presented with a CIN3 lesion 28 months after her last normal Pap smear. This case revealed four, occasionally five, copies of 3q on a diploid background. Interestingly, the subsequent CIN3 lesion showed the same main signal distribution pattern, supporting the hypothesis of clonal expansion. Although the majority of cases that eventually progressed showed increased copy numbers of 3q, a certain percentage of samples contained cells that were tetraploid. When we developed signal number thresholds for the gain of TERC, we therefore included all tetraploid cases. This threshold ensured that all cases that progressed were identified. The sensitivity of our test for predicting progression from CIN1/CIN2 to CIN3 is therefore 100%, and the specificity, that is the prediction of regression, is 70%. These results show that the assay enables us to clearly identify all cervical lesions with a potential of progression. Additionally, the test can identify most women who are at low or no risk of progression. The application of our test to the samples in group 3 also demonstrated the shortcomings of cytological screening: The initial screening assessment in this group revealed normal results in all 12 samples. However, the TERC marker was positive in four of them, which were reviewed by two cytopathologists. The initial diagnosis was upgraded in two of the four cases to CIN2 and CIN3; the other two cases remained “normal.” These cases clearly highlight the potential of the TERC marker: Lesions that are morphologically underdiagnosed can be recognized unambiguously. Biologically, it is of interest that the patterns of aberrations observed in early lesions are usually maintained in higher-grade dysplasias. For instance, one of the samples that was morphologically normal and showed 3q gain had a dominant pattern of 2-3-4 (two signals for CEP7; three signals for CEP3; four signals for TERC) at the time of the first Pap smear in July 1997. The follow-up Pap smear in March 2000 showed the same major signal distribution. At that time, the morphological diagnosis was upgraded to CIN3, and the 2-3-4 pattern was observed in a much higher percentage of cells. This indicates that after the acquisition of a “successful” aneuploid constellation, the cells are fit for clonal expansion, and that the pattern of genomic imbalances then contributes to the genetic make-up of the higher-grade lesion and carcinoma. Another interesting observation was that in a substantial percentage of cases, the gain of 3q developed on a diploid background. A tetraploid intermediate, which has frequently been entertained in the cytogenetic literature of cancer progression, is definitely not a conditio sine qua non for the emergence of chromosomal copy number changes. However, once a tetraploid (4-4-4) pattern is present in a lesion, these cells seem to be “sitting on a fence.” Tetraploid cells can either be eliminated from the population, persist, or can acquire genomic imbalances on the basis of an a priori tetraploid genome, as seen in cases in which we observed a relative gain of 3q in addition to tetraploidy. In conclusion, based on a retrospective analysis of routinely collected cytological samples, we provide evidence that acquisitions of chromosomal aneuploidies that result in a gain of TERC copies are associated with progression of premalignant dysplastic lesions of the uterine cervix. Disease progression in the absence of genomic amplification of TERC was not observed. This test can therefore be used to stratify patients with high specificity and sensitivity. Our data also suggest that the use of this test in conjunction with Pap smears would increase the sensitivity of individual cytological screenings and reduce false-negative diagnoses.
Doubling Up: How a DNA Sequence Becomes a Palindrome
We had previously identified a yeast strain that tolerates DNA palindromes and developed a recombination substrate that produces this class of aberrant product as a readily identifiable byproduct of DNA DSB repair. Provided with the ability to generate dozens of independent palindromes, we developed a method to sequence palindromic DNA in the hope that analysis of the junction at the palindromic center would provide information about the mechanism of palindrome formation. In fact, the structure of the junctions supports a novel view of palindrome origin that involves a foldback and self-priming step. Previously, it was presumed that the junctions were formed by non-homologous end-joining (NHEJ). We demonstrated that palindrome formation is independent of NHEJ functions such as through the Ku complex or DNA ligase IV. We induced a site-specific DSB in a recombination substrate and selected aberrant repair events that disrupted a nearby gene. Physical and genetic assays identified cells with palindromic gene duplications. To sequence the palindromic junctions, we first chemically modified the DNA with sodium bisulfite, which converts cytosine to uracil. This disrupts the intrastrand complementarity of the palindrome, allowing PCR primers (homologous to the modified DNA) to anneal for amplification and sequencing. Alignment of the original starting sequence with the junction sequence indicated that all of the 24 independent junctions analyzed mapped to only 7 sites and shared a characteristic hairpin structure—an example is shown in Figure 1, part A. That is, there was a short hairpin (4–6 bp; shown in red) present at the point of sequence divergence. Furthermore, the novel sequence in the palindromes was precisely complementary to the sequence preceding the first inverted repeat (shown in green). Therefore, the new junction must have been formed by an intramolecular foldback between the 4–6 bp inverted repeats, which in turn served as a primer for new DNA synthesis. Indeed, all 24 junctions fell within a 400 bp region of the target gene and mapped only to sequences capable of forming short hairpins, even though hundreds of other short inverted repeats with longer spacers were present in the same 400 bp. Figure 1. A) Sequence analysis of a palindromic junction: The native sequence prior to DNA double strand break (DSB) induction (top row), the deduced sequence of the palindromic junction obtained by sodium bisulfite sequencing (middle), and the fold-back priming event presumed to have taken place to produce the palindromic junction (bottom). B) Model for intrachromosomal palindrome formation. After the introduction of a DSB, exonucleolytic processing of the ends reveals a short hairpin that can fold back intramolecularly and prime new DNA synthesis. The remaining broken end can invade the other end via short dispersed inverted repeats, leading to break-induced replication (BIR) that proceeds around the newly created hairpin end, thus duplicating the sequence as a palindrome. Holliday junction resolution of the BIR intermediate leads to a long intrachromosomal palindromic duplication. The palindrome can then extrude into a cruciform, triggering a new DSB, initiating a new cycle of amplification (not shown). Gray arrows indicate extent of duplication. Note that if the other broken arm of the chromosome were to experience fold-back priming instead, this would lead to a hairpin-capped chromatid, which upon replication would resemble a sister chromatid fusion event (a common cytological observation in cells undergoing amplification). Cen and tel refer to centromeric and telomeric regions of the chromosome, respectively. A model for the formation of an intrachromosomal palindromic duplication based on our data is shown in Figure 1, part B. Key steps involve the processing of one side of the DSB to reveal a hairpin, followed by fold-back priming and a round of break-induced replication (BIR) of the hairpin into a DNA palindrome. The genetic backgrounds that allow stabilization and recovery of palindromes are cells deficient in any one of the highly conserved genes of the Mre11/Rad50/Xrs2 (MRX) complex (the human ortholog of Xrs2 is Nbs1), or its modulator, Sae2. MRX is required for telomere maintenance, some non-homologous end-joining events, and recovery from cell cycle arrest after treatment of cells with DNA damaging agents (among other roles). Furthermore, MRX is involved in destabilizing palindromes, presumably by the known hairpin cleaving nuclease activity of Mre11. Perhaps a major role of MRX is, in fact, to prevent hairpin fold-back priming from occurring at the site of a broken replication fork. We do not yet know whether the same type of mechanism is operating to create the common palindromic arrays associated with tumorigenesis, but our research opens the door to begin analyzing junctions directly from tumor cells, as well as studying the mechanism and gene requirements for their formation in yeast cells.
Mutations of Thyroid Hormone Nuclear Receptors and Disease
Given the critical roles of TRs in cellular functions, it is reasonable to expect that mutations of TRs could have deleterious consequences. Indeed, shortly after the cloning of the TRβ gene, mutations of it were discovered to cause the genetic syndrome of resistance to thyroid hormone (RTH). However, whether mutations of the TRβ gene cause human diseases other than RTH has been unknown. Likewise, it has been unclear whether mutations of the TRα gene could also cause abnormalities. To address these questions, we used the powerful mouse genetic approach, introducing an identical mutation (PV mutation) into the TRβ and the TRα gene loci. The PV mutation was identified in an RTH patient at the NIH. It is a frame-shift mutation in the C-terminal 16 amino acids of TRβ1 (TRβ1PV) and TRα1 (TRα1PV) that leads to the complete loss of T3 binding activity and transcription capacity (Figure 1). Figure 1. The amino acid sequence of PV and its location in the carboxyl terminus of TRβ1 (A) and TRα1 (B). The PV mutation was identified in a patient with resistance to thyroid hormone. The mutation is from a C-insertion at codon 448 of TRβ1, resulting in a frame-shift mutation in the last 16 carboxyl terminal amino acids. The same PV mutation was targeted to the TRβ and TRα genes to create TRβPV and TRα1PV mice. The knockin mouse that harbors theTRβPV gene (TRβPV mouse; Figure 1, part A) recapitulates human RTH by exhibiting dysregulation of the pituitary-thyroid axis, reduced weight, abnormally accelerated bone development, hypercholesterolemia, and hyperactivity. This mouse model allowed an elucidation of the molecular basis of RTH unattainable otherwise. TRβPV manifests its dominant-negative activity in vivo via competition with wild-type TRs in binding to the promoters of T3-target genes. The variable phenotypic expression in RTH patients is dictated by the tissue-dependent abundance of TR isoforms and modulated by multiple combinatorial cellular factors. Remarkably, homozygous TRβPV/PV mice spontaneously develop follicular thyroid carcinoma, indicating that the deleterious effect of TRβ gene mutations is not limited to RTH. The pathologic progression from hyperplasia to capsular and vascular invasion, and eventually to distant metastasis in TRβPV/PV mice, is similar to human follicular thyroid cancer (Figure 2). Thyroid carcinomas are the most common endocrine neoplasms in humans, with a globally increasing incidence. However, little is known about the molecular genetic events underlying their development. This first mouse model of follicular thyroid carcinoma allows one to discern the genetic alterations contributing to thyroid carcinogenesis and to identify potential molecular targets for prevention and treatment. Indeed, analysis of altered gene expression profiles by cDNA microarray indicates a complex alteration of multiple signaling pathways is associated with thyroid carcinogenesis. One pathway, the peroxisome proliferator–activated receptor γ (PPARγ)–mediated signaling, was found to be repressed by PV during thyroid carcinogenesis. Further molecular study of this pathway led to the identification of PPARγ as a tumor suppressor, thus raising the possibility that PPARγ is a potential target for treatment. Indeed, activation of PPARγ-mediated signaling by treating TRβPV/PV mice with PPARγ agonists significantly delays the development and progression of thyroid carcinogenesis. These results suggest that PV could act as an oncogene by inhibiting the tumor suppressor functions of PPARγ in thyroid carcinogenesis. Figure 2. Morphological features in thyroid glands and metastasis of TRβPV/PV mice. Histological sections from tissues of TRβPV/PV mice stained with hematoxylin (blue) and eosin (pink) show evidence of capsular invasion (A) (arrows) and vascular invasion in thyroid (B) (arrow), spindle cell anaplasia within the thyroid shown at higher magnification (C) (arrow), and a cardiac metastasis (D) (arrow). Capsular and vascular invasion are the pathologic features used in the diagnosis of human neoplastic thyroid tumors. The pathologic progression of thyroid cancer in TRβPV/PV mice is similar to that in humans. The manifestation of the oncogenic actions of PV is not restricted to the thyroid. TRβPV/PV mice also spontaneously develop thyroid stimulating hormone (TSH)–secreting pituitary tumors (TSH-omas). TSH-omas represent about 2% of all pituitary adenomas in humans, affecting vision and causing headaches and other endocrine disorders. The molecular genetics underlying their pathogenesis is largely unknown. Using TRβPV/PV mice as a model, we uncovered a novel mechanism by which PV could function as an oncogene in TSH-omas. PV acts as a constitutive activator of the expression of cyclin D1, a well-known tumor promoter, by tethering to the cyclic AMP response element binding protein (CREB) on the cyclin D1 promoter. These findings suggest that mutation of TRβ is one of the genetic events underlying the pathogenesis of TSH-omas. The knockin mice harboring the PV mutation in the TRα gene (TRα1PV mice; Figure 1, part B) exhibit a phenotype distinct from that of TRβPV mice. Homozygous TRα1PV/PV mice die very shortly after birth. The heterozygous mice (TRα1PV/+) display reduced fertility, increased mortality, delayed bone development, dwarfism, and metabolic disorder, indicating that mutations of TRα lead to severe consequences. The contrasting phenotypes of TRα1PV and TRβPV mice reveal that the actions of TR mutants in vivo are isoform dependent. Analysis of transcription regulation of T3-response genes in several target tissues suggests that distinct phenotypic expression is mediated, in part, by the differentially dominant activity of TR isoform mutants in vivo. The TRα1PV and TRβPV mice have provided a powerful tool to uncover the novel functions of TR mutants and to elucidate their mechanisms of molecular actions in vivo. Accumulated evidence from the study of TRβPV/PV mice suggests that PV, a TRβ mutant, not only causes RTH, but could also function as a new oncogene to contribute to thyroid carcinogenesis and pathogenesis of TSH-omas. Importantly, TRβPV/PV mice could be used as a preclinical model to develop new treatment strategies. Considering that human diseases harboring TRα mutations are yet to be discovered, TRα1PV mice could be used as a model to search for and to identify those diseases whose phenotypic manifestation resembles that of TRα1PV mice.
Scientific Advisory CommitteeIf you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
CCR Frontiers in Science—StaffCenter for Cancer Research Robert H. Wiltrout, PhD, Director Deputy Directors Douglas R. Lowy, MD Editorial Staff Sue Fox, BA/BSW, Senior Editor * Palladian Partners, Inc. FOR INTERNAL USE ONLY |
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iven the current, rapid spread worldwide of HIV-1 among young women of childbearing age, and the effectiveness of antiretroviral drugs to protect the infant from maternal-fetal viral transmission, the number of HIV-1–uninfected children exposed perinatally to highly active antiretroviral therapy (HAART) is likely to increase rapidly. HAART typically includes two nucleoside reverse transcriptase inhibitors (NRTIs), examples of which include zidovudine (AZT), lamivudine (3TC), didanosine (ddI), and stavudine (d4T). These are “dideoxy-type” nucleoside analogs that become phosphorylated and incorporated into viral and host DNA, terminating DNA replication and inducing mutagenesis. Mitochondria are potential sites of toxicity, as NRTIs become incorporated into mitochondrial DNA (mtDNA) and specifically inhibit mitochondrial polymerase γ, causing mtDNA depletion.
ervical cancer screening programs, which are based on the morphological evaluation of Papanicolaou-stained cytological samples (i.e., Pap smears), have greatly reduced the incidence and mortality of cervical cancer in industrialized countries. However, a single cytological evaluation remains relatively insensitive, mainly because of cell sampling from non-representative areas and/or erroneous interpretations. In addition, some early lesions may not have acquired the recognizable phenotypic alterations.
NA palindromes (inverted repeats with little or no spacer) are genetically unstable structures because they can form cruciforms in the DNA, thereby disrupting normal processes such as replication, transcription, and repair. Palindromes are rare in normal cells. However, they are common in tumor cells and are associated with gene amplification. The mechanism by which palindromic gene amplification occurs has been a mystery because the instability of palindromes makes them intractable to molecular studies. In our article, we described the analysis of DNA palindromes generated as an aberrant but fairly common (6%) by-product of DNA double-strand break (DSB) repair in yeast. This detailed analysis has led us to propose a mechanism for how a single copy sequence is initially duplicated to generate a palindrome. 
