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Biomedical subjects

A L Craig

Publications and source records attributed to A L Craig.

7 recordsLinked to original sources
PubMed2001

The human oesophageal squamous epithelium exhibits a novel type of heat shock protein response.

The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.

Amino Acid Sequence↗
PubMed2000

Synergistic activation of p53-dependent transcription by two cooperating damage recognition pathways.

High level activation of p53-dependent transcription occurs following cellular exposure to genotoxic damaging agents such as UV-C, while ionizing radiation damage does not induce a similarly potent induction of p53-dependent gene expression. Reasoning that one of the major differences between UV-C and ionizing radiation damage is that the latter does not inhibit general transcription, we attempted to reconstitute p53-dependent gene expression in ionizing irradiated cells by co-treatment with selected transcription inhibitors that alone do not activate p53. p53-dependent transcription can be dramatically enhanced by the treatment of ionizing irradiated cells with low doses of DRB, which on its own does not induce p53 activity. The mechanism of ionizing radiation-dependent activation of p53-dependent transcription using DRB is more likely due to inhibition of gene transcription rather than prolonged DNA damage, as the non-genotoxic and general transcription inhibitor Roscovitine also synergistically activates p53 function in ionizing irradiated cells. These results identify two distinct signal transduction pathways that cooperate to fully activate p53-dependent gene expression: one responding to lesions induced by ionizing radiation and the second being a kinase pathway that regulates general RNA Polymerase II activity.

Amino Acid Sequence↗
PubMed1999

Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation.

Induction of the transactivation function of p53 after cellular irradiation was studied under conditions in which upstream signaling events modulating p53 activation were uncoupled from those regulating stabilization. This investigation prompted the discovery of a novel radiation-responsive kinase pathway targeting Ser20 that results in the masking of the DO-1 epitope in undamaged cells. Unmasking of the DO-1 epitope via dephosphorylation occurs in response to low doses of non-ionizing radiation. Our data show that phosphorylation at Ser20 reduces binding of the mdm2 protein, suggesting that a function of the Ser20-kinase pathway may be to produce a stable pool of inactive p53 in undamaged cells which can be readily activated after cellular injury. Phospho-specific monoclonal antibodies were used to determine whether the Ser20 signaling pathway is coupled to the Ser15 and Ser392 radiation-responsive kinase pathways. These results demonstrated that: (1) dephosphorylation at Ser20 is co-ordinated with an increased steady-state phosphorylation at Ser392 after irradiation, without p53 protein stabilization, and (2) stabilization of p53 protein can occur without Ser15 phosphorylation at higher doses of radiation. These data show that the Ser20 and Ser392 phosphorylation sites are both targeted by an integrated network of signaling pathways which is acutely sensitive to radiation injury.

Antibodies, Monoclonal↗
PubMed1999

Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers.

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

Amino Acid Sequence↗