RESUMEN
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFß signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores de Tumor/metabolismo , Sistemas CRISPR-Cas/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Ensayos de Selección de Medicamentos Antitumorales , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Indoles , Complejo Mediador/genética , Complejo Mediador/metabolismo , Morfolinas , Neoplasias/genética , Neoplasias/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas , Sulfóxidos/farmacología , Sulfóxidos/uso terapéutico , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
Asunto(s)
Replicación del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Mutaciones Letales Sintéticas , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Humanos , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA2/deficiencia , Resistencia a Antineoplásicos/fisiología , Factor de Transcripción E2F7/fisiología , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/fisiología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Factor de Transcripción E2F7/deficiencia , Técnicas de Inactivación de Genes , Genes BRCA2 , Humanos , Proteínas de Neoplasias/deficiencia , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasas , Recombinasa Rad51/biosíntesis , Recombinasa Rad51/genética , Reparación del ADN por Recombinación/efectos de los fármacos , Reparación del ADN por Recombinación/fisiologíaRESUMEN
OBJECTIVE: Wnt-1-inducible signaling pathway protein 3 (WISP-3)/CCN6 is mutated in progressive pseudorheumatoid dysplasia and may have effects on cartilage homeostasis. The aim of this study was to ascertain additional roles for WISP-3/CCN6 by determining its expression in osteoarthritic (OA) cartilage and by investigating its effects on cartilage-relevant metalloproteinase expression in immortalized (C-28/I2) and primary chondrocytes. METHODS: Cartilage steady-state levels of WISP-3/CCN6 messenger RNA and protein production were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. WISP-3/CCN6 was overexpressed in C-28/I2 cells, and the resultant clones were analyzed by quantitative RT-PCR. The stable clones were analyzed by RT-PCR for metalloproteinase expression, and the signaling pathways involved were investigated using pharmacologic inhibition. The effects of WISP-3/CCN6 on metalloproteinase expression in primary chondrocytes were investigated using a small interfering RNA approach. RESULTS: WISP-3/CCN6 was highly expressed in OA cartilage compared with undamaged cartilage, at both the RNA and protein levels. WISP-3/CCN6 overexpression in C-28/I2 cells resulted in unexpected dual regulation of metalloproteinases; expression of the potent aggrecanase ADAMTS-5 was down-regulated 9-fold, while expression of MMP-10 was up-regulated 14-fold, and these responses were accentuated in the WISP-3/CCN6 clones grown in suspension. MMP-10 up-regulation was dependent on several MAPKs, but WISP-3/CCN6-mediated ADAMTS-5 repression was independent of these pathways and was partially relieved by activation of ß-catenin signaling. WISP-3/CCN6 also suppressed ADAMTS-5 expression in C-28/I2 cells treated with cytokines. In cytokine-treated primary chondrocytes, gene silencing of WISP-3/CCN6 resulted in enhanced ADAMTS-5 expression, while MMP-10 expression was suppressed. CONCLUSION: WISP-3/CCN6 was highly expressed in end-stage OA cartilage, suggesting a role for this growth factor in cartilage homeostasis. WISP-3/CCN6-induced repression of ADAMTS-5 expression and regulation of MMP-10 expression suggest complex and context-dependent roles for WISP-3/CCN6 in cartilage biology.
Asunto(s)
Proteínas CCN de Señalización Intercelular/metabolismo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Metaloproteasas/metabolismo , Osteoartritis de la Rodilla/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas CCN de Señalización Intercelular/genética , Línea Celular , Células Cultivadas , Femenino , Humanos , Masculino , Metaloproteasas/genética , Persona de Mediana Edad , Osteoartritis de la Rodilla/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: Aggrecan is a critical component of cartilage extracellular matrix. Several members of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1ß (IL-1ß) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated. METHODS: MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1ß in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1ß or meniscotibial ligament transaction was carried out in MMP17 null mice. RESULTS: The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1ß mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1ß into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17. CONCLUSION: These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.
Asunto(s)
Agrecanos/metabolismo , Cartílago Articular/metabolismo , Metaloproteinasa 17 de la Matriz/fisiología , Animales , Cartílago Articular/efectos de los fármacos , Endopeptidasas/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Metaloproteinasa 17 de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes/farmacología , Técnicas de Cultivo de TejidosRESUMEN
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Ensayo Cometa , ADN/genética , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Técnica del Anticuerpo Fluorescente , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores , Daño del ADN , Reparación del ADN , Técnicas de Inactivación de Genes , Células HeLa , Recombinación Homóloga/efectos de los fármacos , Humanos , Lisina Acetiltransferasa 5/metabolismo , Proteínas Mad2/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
OBJECTIVE: Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS: Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS: Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION: This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.
Asunto(s)
Artritis Experimental/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Rodilla/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Artritis Experimental/genética , Proteínas CCN de Señalización Intercelular , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Endopeptidasas/biosíntesis , Expresión Génica , Miembro Posterior/patología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/farmacología , Articulaciones/metabolismo , Articulaciones/patología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Metaloproteinasas de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas/genética , Osteoartritis de la Cadera/genética , Osteoartritis de la Rodilla/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/genética , Membrana Sinovial/metabolismoRESUMEN
Apoptosis may play a role in osteoarthritis (OA). Apoptosis can proceed via two different pathways depending on the stimulus. However, both pathways converge upon the effector caspases, caspases-3 and -7. In some systems inhibition of caspases-3 and -7 can prevent apoptosis and may therefore have important therapeutic implications. To confirm this, apoptosis was induced in canine chondrocytes with mitomycin-c (MMC), either in the presence or absence of the general caspase inhibitor, Z-VAD FMK, or a specific caspase-3/7 inhibitor. Z-VAD FMK prevented MMC induced cell death. In contrast, inhibition of caspases-3 and -7 in the presence of MMC induced morphological changes that could be described as necrotic-like or paraptotic-like but did not prevent cell death. The addition of an inhibitor of caspase-8 or caspase-9 along with inhibitor of caspase-3/7 was required to reduce cell death. The morphological changes did not occur in the presence of the caspase-3/7 inhibitor alone and could be prevented by addition of Z-VAD FMK. These data lead to the conclusion that, if the apoptotic program cannot be completed, the cells are pushed into a necrotic or other nonapoptotic mode of death which may involve caspase-8 and/or caspase-9.
Asunto(s)
Inhibidores de Caspasas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mitomicina/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasa 7 , Caspasas/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Condrocitos/enzimología , Perros , Microscopía Electrónica de Transmisión , FenotipoRESUMEN
UNLABELLED: Over 21 days in culture, cell death spreads, both radially and transversely, from loaded to surrounding cartilage. This spread was prevented by physical separation and separate culture post-impact. OBJECTIVE: One aim was to determine if nitric oxide (NO) is the intercellular signal mediating cell death. Another aim was to clarify the nature of the cell death, whether caspase mediated apoptosis or necrosis. DESIGN: Cyclic impacts were applied to the central 2 mm core of 4 mm canine articular cartilage discs. Post-impact culturing was for 21 days in the presence or absence of the iNOS inhibitor, L-NAME, or the broad-spectrum caspase inhibitor, Z-VAD FMK. Cell death was quantified using the TUNEL assay. Culture media were collected every 2 days for measurements of glycosaminoglycan (GAG) and NO release. RESULTS: Cell death spread from the loaded core into the surrounding ring over 21 days in culture. Although L-NAME significantly reduced nitrite release into the culture media of both loaded and control cartilage, the spread of cell death was not prevented. Neither was the spread of cell death prevented by Z-VAD FMK. CONCLUSIONS: These data indicate that NO is not acting as an intercellular signalling factor in this in vitro system and that the cell death post-impact is not caspase mediated.
Asunto(s)
Cartílago Articular/fisiología , Caspasas/metabolismo , Muerte Celular/fisiología , Óxido Nítrico/fisiología , Clorometilcetonas de Aminoácidos/antagonistas & inhibidores , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Apoptosis/fisiología , Cartílago Articular/lesiones , Cartílago Articular/patología , Células Cultivadas , Medios de Cultivo , Inhibidores de Cisteína Proteinasa/metabolismo , Perros , Inhibidores Enzimáticos/metabolismo , Glicosaminoglicanos/análisis , Etiquetado Corte-Fin in Situ , NG-Nitroarginina Metil Éster/metabolismo , Necrosis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Nitritos/análisisRESUMEN
We sought to explain previously reported associations between osteoarthritis, cartilage collagen denaturation, and repetitive mechanical loading. Eighty specimens of cartilage-on-bone were obtained from bovine patella grooves. After soaking in protease inhibitors, specimens were subjected to 3,600 cycles of compressive loading by means of a flat indenter. Loaded cartilage was supported by adjacent cartilage and subchondral bone. Peak stress ranged between 3.5 MPa and 14 MPa. Collagen denaturation was assessed by an inhibition ELISA assay using the col2-3/4 m antibody. Results showed that denaturation increased with loading severity, from 2% in control (unloaded) cartilage to 7.5% at 14 MPa (p < .0003). Collagen and glycosaminoglycan content of cartilage were unchanged after loading. Denaturation was largely unaffected by the absence of protease inhibitors or when chondrocytes were killed by repeated freeze-thaw cycles prior to loading. We conclude that vigorous cyclic mechanical loading causes an immediate dose-related increase in collagen denaturation in bovine articular cartilage.
Asunto(s)
Cartílago Articular/metabolismo , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Soporte de Peso/fisiología , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Desnaturalización Proteica/fisiologíaRESUMEN
OBJECTIVE: To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin-1beta (IL-1beta), IL-1beta-converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted. METHODS: Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild-type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0-6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti-VDIPEN, anti-NITEGE, and Col2-3/4C(short) neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique. RESULTS: All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild-type mice. ICE-, iNOS-, and particularly IL-1beta-knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP-, aggrecanase-, and collagenase-generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions. CONCLUSION: We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.