Biomedical subjects

J C Sible

Publications and source records attributed to J C Sible.

7 recordsLinked to original sources

PubMed2000

Dissection of the XChk1 signaling pathway in Xenopus laevis embryos.

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints, xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 in Xenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.

Animals↗

PubMed1998

Developmental regulation of MCM replication factors in Xenopus laevis.

At the midblastula transition (MBT) during Xenopus laevis development, zygotic transcription begins [1], and the rapid, early cleavage cycles are replaced by cell-division cycles that lengthen and acquire G (gap) phases [2] and checkpoints [3-5]. This cell-cycle remodeling may result from either a loss of maternal products, the transcription of zygotic genes, or the replacement of maternal proteins by zygotic gene products. We have identified an example of the third possibility: distinct maternal and zygotic genes encoding a member of the minichromosome maintenance (MCM) protein family. The mcm genes were identified in yeast by mutations that blocked replication of artificial chromosomes or perturbed the G1/S transition in the cell cycle [6,7]. In Xenopus eggs, the MCM2-MCM7 proteins assemble as multimeric complexes at chromosomal origins of replication [8-14]. The sequential, cell-cycle-dependent assembly of the origin replication complex (ORC), CDC6 protein and the MCM complex at origins of replication ensures that DNA replicates only once per cell cycle [15,16]. The periodic association of the MCM complex with chromatin may be regulated via phosphorylation by cyclin-dependent kinases (Cdks) [11]. We have cloned the first example of a developmentally regulated mcm gene, zygotic mcm6 (zmcm6), expressed only after gastrulation when the cell cycle is remodeled. The zMCM6 protein assembles into MCM complexes and differs from maternal MCM6 (mMCM6) in having a carboxy-terminal extension and a consensus cyclin-Cdk phosphorylation site. There may also be maternal-zygotic pairs of other MCMs. These data suggest that MCMs are critical for cell-cycle remodeling during early Xenopus development.

Animals↗

PubMed1997

Zygotic transcription is required to block a maternal program of apoptosis in Xenopus embryos.

At the midblastula transition during Xenopus development, the cell cycle is remodeled, and zygotic transcription is initiated. Additionally, cyclin E1 is degraded at the midblastula transition independently of protein synthesis, the number of cell cycles, and the nuclear-to-cytoplasmic ratio. In the studies reported here, cell cycles were delayed by transient inhibition of protein synthesis with cycloheximide (100 microg/ml) prior to the midblastula transition. Even after reaccumulation of mitotic cyclins and resumption of cell divisions, cycloheximide-treated embryos did not resume DNA synthesis, failed to initiate transcription, and synchronously became apoptotic before the gastrula stage. These results were independent of the stage at which embryos were treated or the duration of treatment. Inhibition of zygotic transcription with alpha-amanitin also induced apoptosis. These data suggest that a developmental checkpoint at the midblastula transition is maternally regulated and can trigger apoptosis. Apoptosis induced by cycloheximide or alpha-amanitin was blocked by injection of RNA encoding Xenopus Bcl-2, suggesting that this maternal program is normally blocked by expression of an apoptotic inhibitor. Embryos pulsed with lower doses of cycloheximide (10 microg/ml) delayed development prior to the midblastula transition but resumed DNA synthesis, initiated transcription, and gastrulated normally. This indicates that the apoptotic response is initiated only when delayed embryos are unable to support initiation of zygotic transcription.

Amanitins↗

PubMed1997

A role for cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos.

During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.

Animals↗

PubMed1996

DNA binding proteins from keloid fibroblasts form unique complexes with the human fibronectin promoter.

Keloids are pathological lesions characterized by enhanced expression of extracellular matrix molecules including fibronectin. A molecular dissection of the human fibronectin promoter was performed to identify DNA-protein interactions that correlate with altered fibronectin gene expression by keloid fibroblasts. DNA mobility shift patterns generated by nuclear extracts from skin, scar, and keloid fibroblasts were identical at a consensus CRE at -170 of the human fibronectin promoter whereas extracts from keloid fibroblasts formed complexes at a CRE/AP-1-like sequence at -415 that differed from those generated by skin and scar fibroblast extracts. The DNA-protein interactions identified at -415 were sensitive to altered serum concentrations in skin and scar but not keloid fibroblast cultures. The effects of forskolin and TGF-beta on fibronectin expression correlated with changes in the DNA-protein complexes assembled on the -170 and -415 cis elements, respectively. Oligonucleotides containing consensus CRE and AP-1 sequences did not compete for binding of nuclear proteins to the CRE/AP-1-like domain at -415, suggesting that this is a unique cis element. These studies indicate that the human fibronectin promoter contains two cis elements on which related but nonidentical complexes form. Alterations in the complexes interacting with the sequence at -415 may be responsible for the differences in fibronectin gene expression among quiescent skin, mature scar, and keloid fibroblasts.

Adolescent↗

PubMed1994

Fibronectin gene expression differs in normal and abnormal human wound healing.

The overproduction of fibronectin and type I collagen in keloids and hypertrophic scars implicates altered regulation of extracellular matrix components as an important aspect of these wound healing pathologies. However, little is known about the similarities and differences in extracellular matrix gene expression during normal and abnormal wound healing. This study compared the content of fibronectin messenger RNA and rates of fibronectin protein biosynthesis in fibroblasts derived from normal skin, normal scar, keloid, and hypertrophic scar. Fibronectin expression was enhanced in cells from both normal and abnormal wounds relative to cells from quiescent normal skin. Matched pairs of normal and keloid fibroblasts from the same individuals were also compared, and three of the four pairs showed higher fibronectin expression by the keloid cells at the levels of messenger RNA and protein synthesis. This was consistent with previous studies showing elevated steady state content of fibronectin in keloid cells relative to normal cells from the same individual. Fibronectin messenger RNA and protein content in the tissues from which these cells were derived was examined by in situ hybridization and immunohistochemistry. These studies revealed that in vivo, the steady state content of fibronectin messenger RNA and protein was highest in abnormal wounds, less in most normal scars, and lowest in normal skin. Thus, fibroblasts from keloids and hypertrophic scars overexpressed fibronectin in vivo relative to normal skin and normal scar and retain this characteristic in vitro relative to normal skin. Although normal scars contained little fibronectin protein and messenger RNA, cultured fibroblasts derived from these scars had contents of fibronectin messenger RNA and rates of biosynthesis in vitro similar to those of keloid fibroblasts. This indicates that the fibronectin regulatory pathway in scar fibroblasts is influenced by the tissue environment. These results are discussed with respect to the relationship of fibronectin expression in keloids, hypertrophic scars, and normal wounds in human beings.

Journal Article↗

PubMedDate not supplied

Gene expression of tenascin is altered in normal scars and keloids.

Tenascin is an extracellular matrix molecule with structural similarity to fibronectin. An increase in extracellular matrix content of both tenascin and fibronectin is associated with early wound healing and with various skin fibroses. However, the relationship of tenascin and fibronectin expression during scar remodeling and the formation of pathologic scars such as keloids is unknown. Expression of tenascin in normal and abnormal human scars was examined and compared with that of fibronectin by immunohistochemistry and in situ hybridization. Tenascin and fibronectin protein and messenger RNA contents were elevated in normal, mature scars relative to quiescent skin, similar to the situation during earlier stages of healing. Tenascin and fibronectin expression was further enhanced in keloids relative to normal skin and scar, and, as has been shown for fibronectin, tenascin expression in uninjured skin adjacent to keloids was indistinguishable from that in quiescent skin from unaffected individuals. These data suggest that tenascin and fibronectin gene expression are coordinated during later stages of normal wound healing and that a defect involving common regulatory elements for these genes is associated with the formation of keloids.

Journal Article↗