RESUMEN
The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.
Asunto(s)
Calcio , Citosol , Replicación del ADN , Proteínas de la Membrana , Canales Catiónicos TRPV , Humanos , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Calcio/metabolismo , Citosol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Células HEK293 , ADN/metabolismo , Células HeLa , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Fosforilación , Inestabilidad Genómica , Daño del ADN , AnimalesRESUMEN
Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA 1 . However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC) 2 . In addition to its established binding to N 6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N 1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These R NA d amage-induced DNA b reaks (RDIBs) depend on R-loop formation, which in turn are processed by factors involved in transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role for YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.
RESUMEN
The central dogma of molecular biology posits that genetic information flows unidirectionally, from DNA, to RNA, and finally to protein. However, this directionality is broken in some cases, such as reverse transcription where RNA is converted to DNA by retroviruses and certain transposable elements. Our genomes have evolved and adapted to the presence of reverse transcription. Similarly, our genome is continuously maintained by several repair pathways to reverse damage due to various endogenous and exogenous sources. More recently, evidence has revealed that RNA, while in certain contexts may be detrimental for genome stability, is involved in promoting certain types of DNA repair. Depending on the pathway in question, the size of these DNA repair-associated RNAs range from one or a few ribonucleotides to long fragments of RNA. Moreover, RNA is highly modified, and RNA modifications have been revealed to be functionally associated with specific DNA repair pathways. In this review, we highlight aspects of this unexpected layer of genomic maintenance, demonstrating how RNA may influence DNA integrity.
Asunto(s)
Daño del ADN , ARN , Humanos , ARN/genética , Reparación del ADN , ADN/metabolismo , Proteínas , Inestabilidad GenómicaRESUMEN
The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.
Asunto(s)
Síndromes de Tricotiodistrofia , Animales , Ratones , Intrones/genética , Síndromes de Tricotiodistrofia/genética , ARN Nucleotidiltransferasas/genética , Empalme del ARNRESUMEN
Activating signal co-integrator 1 complex (ASCC) subunit 3 (ASCC3) supports diverse genome maintenance and gene expression processes, and contains tandem Ski2-like NTPase/helicase cassettes crucial for these functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. We present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. TRIP4 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning an ASC-1 homology domain next to the C-terminal helicase cassette of ASCC3, likely supporting substrate engagement and assisting the DNA exit. TRIP4 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC3 for specific processes. Our findings define ASCC3-TRIP4 as a tunable motor module of ASCC that encompasses two cooperating NTPase/helicase units functionally expanded by TRIP4.
Asunto(s)
ADN Helicasas , Nucleósido-Trifosfatasa , Nucleósido-Trifosfatasa/metabolismo , ADN Helicasas/metabolismo , Empalmosomas/metabolismo , ARN Helicasas/metabolismo , ADN/metabolismoRESUMEN
Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.
Asunto(s)
Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Alquilación , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Metilación , Fosforilación , Procesamiento Proteico-Postraduccional , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genéticaRESUMEN
Cellular RNAs are modified by both physiological factors and exogenous agents, such as methyl methanesulfonate (MMS). However, techniques for analyzing how proteins may interact with these modified RNAs are limited. Here, we provide a protocol combining RNA immunoprecipitation (RIP) with mass spectrometry (MS) to analyze the methylation state of the RNAs bound by Flag-tagged proteins in mammalian cells. The approach is highly quantitative and can simultaneously detect several methylated nucleosides in a single experiment. For complete details on the use and execution of this protocol, please refer to Tsao et al. (2021).
Asunto(s)
Nucleósidos , ARN , Animales , Inmunoprecipitación , Mamíferos/genética , Espectrometría de Masas/métodos , Metilación , Nucleósidos/análisis , Proteínas , ARN/químicaRESUMEN
Physiological and chemically induced modifications to nucleosides are common in both DNA and RNA. Physiological forms of these modifications play critical roles in gene expression, yet aberrant marks, if left unrepaired, may be associated with increased genome instability. Due to the low prevalence of these marks in most samples of interest, a highly sensitive method is needed for their detection and quantitation. High-performance liquid chromatography, coupled to mass spectrometry (HPLC-MS), provides this high degree of sensitivity while also being adaptable to nearly any modified nucleoside of interest and still maintaining exquisite specificity. In this chapter, we demonstrate how to use HPLC-MS to analyze the catalytic activity of a nucleic acid demethylase, to quantify the prevalence of N6-methyladenosine from RNA, and to determine the kinetics of alkylation damage repair.
Asunto(s)
Nucleósidos , ARN , Cromatografía Líquida de Alta Presión/métodos , ADN , Espectrometría de Masas , Nucleósidos/químicaRESUMEN
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Núcleo Celular/enzimología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias/enzimología , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Núcleo Celular/genética , ADN Helicasas/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Metilación , Neoplasias/genética , Proteínas Nucleares/genética , Estructuras R-Loop , ARN Neoplásico/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Transcripción Genética , UbiquitinaciónRESUMEN
DNA damaging agents have been a cornerstone of cancer therapy for nearly a century. The discovery of many of these chemicals, particularly the alkylating agents, are deeply entwined with the development of poisonous materials originally intended for use in warfare. Over the last decades, their anti-proliferative effects have focused on the specific mechanisms by which they damage DNA, and the factors involved in the repair of such damage. Due to the variety of aberrant adducts created even for the simplest alkylating agents, numerous pathways of repair are engaged as a defense against this damage. More recent work has underscored the role of RNA damage in the cellular response to these agents, although the understanding of their role in relation to established DNA repair pathways is still in its infancy. In this review, we discuss the chemistry of alkylating agents, the numerous ways in which they damage nucleic acids, as well as the specific DNA and RNA repair pathways which are engaged to counter their effects.
Asunto(s)
Daño del ADN , ADN/genética , ARN/genética , Alquilantes/toxicidad , Alquilación/efectos de los fármacos , Animales , ADN/química , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Humanos , ARN/químicaRESUMEN
NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.
Asunto(s)
Daño del ADN , Mitocondrias/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Mitocondrias/genética , Nucleósido Difosfato Quinasas NM23/genéticaRESUMEN
Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Reparación del ADN , Lisina Acetiltransferasa 5/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Células HEK293 , Células HeLa , Humanos , Lisina Acetiltransferasa 5/genética , Complejos Multienzimáticos/genética , Nucleósido-Fosfato Quinasa/genética , Fosforilación/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismoRESUMEN
The appropriate supply of dNTPs is critical for cell growth and genome integrity. Here, we investigated the interrelationship between dUTP pyrophosphatase (dUTPase) and ribonucleotide reductase (RNR) in the regulation of genome stability. Our results demonstrate that reducing the expression of dUTPase increases genome stress in cancer. Analysis of clinical samples reveals a significant correlation between the combination of low dUTPase and high R2, a subunit of RNR, and a poor prognosis in colorectal and breast cancer patients. Furthermore, overexpression of R2 in non-tumorigenic cells progressively increases genome stress, promoting transformation. These cells display alterations in replication fork progression, elevated genomic uracil, and breaks at AT-rich common fragile sites. Consistently, overexpression of dUTPase abolishes R2-induced genome instability. Thus, the expression level of dUTPase determines the role of high R2 in driving genome instability in cancer cells.
Asunto(s)
Inestabilidad Genómica/genética , Neoplasias/genética , Pirofosfatasas/genética , Ribonucleótido Reductasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Sitios Frágiles del Cromosoma/genética , Femenino , Células HT29 , Humanos , Células MCF-7 , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs.
Asunto(s)
Reparación del ADN/fisiología , Histona Acetiltransferasas/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Ribonucleótido Reductasas/metabolismo , Células HeLa , Histona Acetiltransferasas/genética , Humanos , Lisina Acetiltransferasa 5 , Células MCF-7 , Nucleósido Difosfato Quinasas NM23/genética , Ribonucleótido Reductasas/genéticaRESUMEN
Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.
Asunto(s)
Reparación del ADN , Nucleósido-Fosfato Quinasa/metabolismo , Medio de Cultivo Libre de Suero , Citidina/metabolismo , Daño del ADN , Reparación del ADN/efectos de la radiación , Nucleótidos de Desoxicitosina/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Rayos UltravioletaRESUMEN
(99m)Tc-N4-guanine ((99m)Tc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of (99m)Tc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using (99m)Tc-N4amG in tumor specific imaging.
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Neoplasias Mamarias Experimentales/metabolismo , Compuestos de Organotecnecio/síntesis química , Compuestos de Organotecnecio/farmacocinética , Animales , Femenino , Espectroscopía de Resonancia Magnética , Dosis de Radiación , Ratas , Ratas Endogámicas F344 , Distribución TisularRESUMEN
The synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.
Asunto(s)
Reparación del ADN , Nucleótidos de Desoxiuracil/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Daño del ADN , Doxorrubicina/farmacología , Femenino , Ratones , Ratones Endogámicos BALB C , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismoRESUMEN
Activation of hedgehog (HH) pathway signaling is observed in many tumors. Due to a feedback loop, the HH receptor Patched (PTCH-1) is overexpressed in tumors with activated HH signaling. Therefore, we sought to radiolabel the PTCH-1 ligand sonic (SHH) for detection of cancer cells with canonical HH activity. Receptor binding of ¹³¹I-SHH was increased in cell lines with high HH pathway activation. Our findings also show that PTCH-1 receptor expression is decreased upon treatment with HH signaling inhibitors, and receptor binding of ¹³¹I-SHH is significantly decreased following treatment with cyclopamine. In vivo imaging and biodistribution studies revealed significant accumulation of ¹³¹I-SHH within tumor tissue as compared to normal organs. Tumor-to-muscle ratios were approximately 8 : 1 at 5 hours, while tumor to blood and tumor to bone were 2 : 1 and 5 : 1, respectively. Significant uptake was also observed in liver and gastrointestinal tissue. These studies show that ¹³¹I-SHH is capable of in vivo detection of breast tumors with high HH signaling. We further demonstrate that the hedgehog receptor PTCH-1 is downregulated upon treatment with hedgehog inhibitors. Our data suggests that radiolabeled SHH derivatives may provide a method to determine response to SHH-targeted therapies.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Hedgehog/metabolismo , Marcaje Isotópico , Transducción de Señal , Animales , Bioensayo , Western Blotting , Neoplasias de la Mama/diagnóstico por imagen , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Cámaras gamma , Humanos , Radioisótopos de Yodo , Proteínas Oncogénicas/metabolismo , Receptores Patched , Receptor Patched-1 , Unión Proteica/efectos de los fármacos , Radiometría , Cintigrafía , Ratas , Ratas Endogámicas F344 , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Distribución Tisular/efectos de los fármacos , Transactivadores/metabolismo , Alcaloides de Veratrum/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1RESUMEN
Objective. This study was aimed to study tissue distribution and tumor imaging potential of (68)Ga-glycopeptide (GP) in tumor-bearing rodents by PET. Methods. GP was synthesized by conjugating glutamate peptide and chitosan. GP was labeled with (68)Ga chloride for in vitro and in vivo studies. Computer outlined region of interest (counts per pixel) of the tumor and muscle (at the symmetric site) was used to determine tumor-to-muscle count density ratios. To ascertain the feasibility of (68)Ga-GP in tumor imaging in large animals, PET/CT imaging of (68)Ga-GP and (18)F-FDG were conducted in New Zealand white rabbits bearing VX2 tumors. Standard uptake value of tumors were determined by PET up to 45 min. To determine blood clearance and half-life of (68)Ga-GP, blood samples were collected from 10 seconds to 20 min. Results. Radiochemical purity of (68)Ga-GP determined by instant thin-layer chromatography was >95%. Tumor uptake values (SUV) for (68)Ga-GP and (18)F-FDG in New Zealand white rabbits bearing VX2 tumors were 3.25 versus 7.04. PET images in tumor-bearing rats and rabbits confirmed that (68)Ga-GP could assess tumor uptake. From blood clearance curve, the half-life of (68)Ga-GP was 1.84 hr. Conclusion Our data indicate that it is feasible to use (68)Ga-GP to assess tumor angiogenesis.
Asunto(s)
Glicopéptidos , Neoplasias/irrigación sanguínea , Neovascularización Patológica/diagnóstico por imagen , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos X , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/diagnóstico por imagen , Cromatografía en Gel , Relación Dosis-Respuesta en la Radiación , Electroforesis Capilar , Femenino , Radioisótopos de Galio , Glicopéptidos/sangre , Glicopéptidos/química , Glicopéptidos/farmacocinética , Semivida , Neoplasias/diagnóstico por imagen , Conejos , Ratas , Ratas Endogámicas F344 , Distribución TisularRESUMEN
OBJECTIVE: This study was to develop a (99m)Tc-labeled alpha-methyl tyrosine (AMT) using L,L-ethylenedicysteine (EC) as a chelator and to evaluate its potential in breast tumor imaging in rodents. METHODS: EC-AMT was synthesized by reacting EC and 3-bromopropyl AMT (N-BOC, ethyl ester) in ethanol/potassium carbonate solution. EC-AMT was labeled with (99m)Tc in the presence of tin (II) chloride. Rhenium-EC-AMT (Re-EC-AMT) was synthesized as a reference standard for (99m)Tc-EC-AMT. To assess the cellular uptake kinetics of (99m)Tc-EC-AMT, 13 762 rat breast cancer cells were incubated with (99m)Tc-EC-AMT for 0-2 h. To investigate the transport mechanism, the same cell line was used to conduct the competitive inhibition study using L-tyrosine. Tissue distribution of (99m)Tc-EC-AMT was determined in normal rats at 0.5-4 h. Planar imaging of breast tumor-bearing rats was performed at 30 and 90 min. The data were compared with those of (18)F-2-fluoro-2-deoxy-glucose. Blocking uptake study using unlabeled AMT was conducted to investigate the transport mechanism of (99m)Tc-EC-AMT in vivo. RESULTS: Structures of EC-AMT and Re-EC-AMT were confirmed by nuclear magnetic resonance, high performance liquid chromatography and mass spectra. In-vitro cellular uptake of (99m)Tc-EC-AMT in 13,762 cells was increased as compared with that of (99m)Tc-EC and could be inhibited by L-tyrosine. Biodistribution in normal rats showed high in-vivo stability of (99m)Tc-EC-AMT. Planar scintigraphy at 30 and 90 min showed that (99m)Tc-EC-AMT could clearly visualize tumors. (99m)Tc-EC-AMT uptake could be significantly blocked by unlabeled AMT in vivo. CONCLUSION: The results indicate that (99m)Tc-EC-AMT, a new amino acid transporter-based radiotracer, is suitable for breast tumor imaging.