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T R Hupp

Publications and source records attributed to T R Hupp.

At least 19 recordsLinked to original sources
PubMed2006

HIF-1alpha contributes to tumour-selective killing by the sigma receptor antagonist rimcazole.

We have previously reported tumour-selective killing by the sigma (sigma) receptor ligand rimcazole. We now report that rimcazole elevates hypoxia inducible factor-1alpha (HIF-1alpha) protein levels under normoxic conditions in colorectal (HCT-116) and mammary carcinoma (MDA MB 231) cells but fails to induce HIF-1alpha in normal fibroblasts or mammary epithelial cells. Combining the sigma-1 agonist (+)-pentazocine with rimcazole substantially reduces the accumulation of HIF-1alpha, confirming that the effect is mediated at least partly by antagonism of sigma-1 sites. HIF-1alpha knockdown by RNA interference attenuates rimcazole-induced cell death in both cell types. Thus, the induction of HIF-1alpha by rimcazole contributes to tumour cell killing. In a comparison of HCT-116p53+/+ and HCT-116p53-/- cells, HIF-1alpha levels are consistently higher after rimcazole treatment in HCT-116p53+/+ cells. Furthermore, although rimcazole kills HCT-116p53-/- cells, it has a more potent apoptosis-inducing effect in HCT-116p53+/+ cells. This suggests that the presence of functional p53 protein may enhance death induction by rimcazole in part through greater induction of HIF-1alpha. p53 is not required, however, for the rimcazole-induced engagement of HIF-1alpha in proapoptotic mode as HIF-1alpha knockdown attenuates rimcazole-induced death to comparable extents in p53 mutant and wild-type cell systems. Knowledge of HIF-1alpha involvement may assist the re-profiling of rimcazole and other sigma ligands as cancer therapeutics.

Apoptosis↗
PubMed2006

Epidermal stem cells and cancer stem cells: insights into cancer and potential therapeutic strategies.

Epithelial keratinocyte regeneration has been exemplified as dependent on a population of cellular progenitors that have retained developmental pluripotency, a latent capacity for proliferation and differentiation with a prolonged lifespan. Recent evidence suggests that the cell populations that regulate the development of normal tissues, and which play vital roles in maintaining the overall homeostasis of the tissue, might be the key target population that is essential for malignant cancer development, thus giving rise to the notion of 'cancer stem cells'. This review examines the leading research into the relationship between adult stem cells in human skin marked by p63alphaDeltaN, their putative importance in cancer development, and how we might exploit our evolving knowledge of adult tissue stem cells to aid cancer treatments in the future. Furthermore, the review examines information regarding ataxia telangiectasia mutated (ATM) kinase and key regulatory events that take place on p53, only within putative keratinocyte stem cells that are transcriptionally regulated by p63alphaDeltaN.

Cell Proliferation↗
PubMed2005

The effect of ultraviolet (UV) A1, UVB and solar-simulated radiation on p53 activation and p21.

BACKGROUND: High-dose ultraviolet (UV) A1 therapy (doses in the order of 130 J cm(-2)) is effective for atopic dermatitis and scleroderma. UVA1 has been shown to induce a dose-dependent increase in p53 expression in keratinocytes. OBJECTIVES: To examine the effect of UVA1 on the activation of p53 by phosphorylation, which has not yet been studied. METHODS: Five adult volunteers were exposed to dose series of UVA1 (10-100 J cm(-2)) and, for comparison, narrowband UVB (TL-01) (25-550 mJ cm(-2)) and solar-simulated radiation (SSR) (5.6-30 J cm(-2)) on photoprotected buttock skin and the minimal erythema dose (MED) for each was determined at 24 h. Separate sites on the buttock were subsequently irradiated with a 3-MED dose of UVA1, TL-01 and SSR. At 24 h, punch biopsies (4 mm) were taken from each irradiated site and from an adjacent unirradiated control site, and immunohistochemical staining for p53 (Do-1), activation of p53 (assessed by phosphorylation at serine 15 and serine 392) and p21 was performed. Cell staining was expressed as the mean number of cells stained per three high-power fields (HPFs) and as a percentage of 1000 cells. Sunburn cells (SBCs) were also counted per HPF. RESULTS: UVA1 produced negligible numbers of SBCs, relatively little p53 (Do-1) staining (mean +/- SD cell count per HPF 16 +/- 10), no p53 activation and very little evidence of p21 expression (mean +/- SD cell count per HPF 5.3 +/- 7), in contrast to TL-01 (mean +/- SD cell count per HPF of 11.83 +/- 2.1 SBCs, 146.3 +/- 38 for Do-1, 26.6 +/- 15 for serine 15, 14.9 +/- 12 for serine 392 and 77.9 +/- 30 for p21) or SSR irradiation (mean +/- SD cell count per HPF of 3.5 +/- 1.2 SBCs, 147.5 +/- 62 for Do-1, 54 +/- 50 for serine 15, 38.9 +/- 18 for serine 392 and 56.7 +/- 30 for p21). CONCLUSIONS: These data indicate that there are fundamental differences in the effects of UVA1 on p53 and its activation pathways compared with TL-01 and SSR, and may in part explain the differential effects of these phototherapies.

Adult↗
PubMed2005

Differential effects of 5-aminolaevulinic acid photodynamic therapy and psoralen + ultraviolet A therapy on p53 phosphorylation in normal human skin in vivo.

BACKGROUND: Phosphorylation of the tumour suppressor p53 by the CK2/FACT pathway plays a central role in suppressing ultraviolet (UV)-induced skin cancer in animal models. Although p53 protein stabilization is induced after solar-simulated irradiation of human skin in vivo, p53 phosphorylation has not been defined. OBJECTIVES: To investigate the effects of clinically effective treatments for skin diseases including psoralen + UVA (PUVA) and photodynamic therapy (PDT) on p53 phosphorylation to determine whether the tumour-suppressing p53 kinase pathways are activated upon use of these therapies. METHODS: We used antibodies to the ATM/ATR and CK2/FACT phosphorylation sites on p53. RESULTS: We found that p53 activation was induced selectively by PUVA treatment, while 8-oxo-7,8-dihydroguanine DNA damage was induced selectively by 5-aminolaevulinic acid (ALA)-PDT treatment. Importantly, PUVA treatment resulted in p53 kinase activation, as defined by p53 modification at AT (serine-15) and CK2/FACT (serine-392) sites within the proliferative compartment. CONCLUSIONS: These data demonstrate that PUVA provokes accumulation and phosphorylation of p53 by AT and CK2/FACT within critical proliferative focal points (as determined by p63 colocalization studies) where DNA damage may lead to tumorigenesis. PDT is mechanistically distinct in that there is a lower level of induction of p53 expression with no evidence of AT- or CK2/FACT-mediated phosphorylation. This suggests that the type of DNA damage created by the reactive oxygen species generated by ALA-PDT does not induce the p53 pathway classically required for the repair of DNA photoadducts induced by UV.

Aminolevulinic Acid↗
PubMed2003

Doxorubicin and vinorelbine act independently via p53 expression and p38 activation respectively in breast cancer cell lines.

In the treatment of breast cancer, combination chemotherapy is used to overcome drug resistance. Combining doxorubicin and vinorelbine in the treatment of patients with metastatic breast cancer has shown high response rates; even single-agent vinorelbine in patients previously exposed to anthracyclines results in significant remission. Alterations in protein kinase-mediated signal transduction and p53 mutations may play a role in drug resistance with cross-talk between signal transduction and p53 pathways. The aim of this study was to establish the effects of doxorubicin and vinorelbine, as single agents, in combination, and as sequential treatments, on signal transduction and p53 in the breast cancer cell lines MCF-7 and MDA-MB-468. In both cell lines, increased p38 activity was demonstrated following vinorelbine but not doxorubicin treatment, whether vinorelbine was given prior to or simultaneously with doxorubicin. Mitogen-activated protein kinase (MAPK) activity and p53 expression remained unchanged following vinorelbine treatment. Doxorubicin treatment resulted in increased p53 expression, without changes in MAPK or p38 activity. These findings suggest that the effect of doxorubicin and vinorelbine used in combination may be achieved at least in part through distinct mechanisms. This additivism, where doxorubicin acts via p53 expression and vinorelbine through p38 activation, may contribute to the high clinical response rate when the two drugs are used together in the treatment of breast cancer.

Breast Neoplasms↗
PubMed2001

Expression of the Hypermethylated in Cancer gene (HIC-1) is associated with good outcome in human breast cancer.

A new cancer gene, HIC-1 (Hypermethylated in Cancer) telomeric to p53 on chromosome 17p may be of clinical importance in sporadic breast cancer. Regional DNA hypermethylation of 17p13.3 resulting in suppression of gene expression has been shown to precede 17p structural changes in human carcinogenesis. In addition, loss of heterozygosity studies have suggested clinically significant involvement of a gene on 17p13.3 associated with poor prognosis in breast cancer. Using RT-PCR analysis, we demonstrate that the MCF7 (wild type p53) cell line expressed HIC-1 transcripts but the MDAMB231 (mutant p53) cell line did not, suggesting loss of HIC-1 expression and p53 malfunction may be synergistic events in sporadic breast cancer. HIC-1 expression was examined using RT-PCR on RNA extracted from 50 primary untreated, human breast cancers and was detected in only 7/50 (14%) cancers. All seven patients with HIC-1 expression were alive without disease recurrence after 8 years follow-up and 5/7 had detectable p53 wild type mRNA expression. This suggests that retained HIC-1 expression may offer a survival advantage. However the seven cancers had 17p13.3 loss of heterozygosity (LOH; four patients), a feature previously associated with poor prognosis, or were homozygous (three patients) suggesting there may be two genes at 17p13.3 involved in breast carcinogenesis. Using a demethylating drug 5-aza-2'-deoxycytidine (DeoxyC), HIC-1 expression was restored in the MDAMB231 cells, also suggesting restoration of HIC-1 function by reversing HIC-1 hypermethylation may offer a therapeutic avenue in breast cancer.

Antimetabolites, Antineoplastic↗
PubMed2001

Differential p53 protein expression in breast cancer fine needle aspirates: the potential for in vivo monitoring.

Fine needle aspiration (FNA) biopsy is the least invasive method of sampling breast cancer in vivo and provides material for breast cancer diagnosis. FNA has also been used to examine cellular markers to predict and monitor the effects of therapy. The aim of this study was to assess the accuracy of using FNA material compared with resected cancer for Western blotting studies of the p53 pathway, a key to tumour response to radiotherapy and chemotherapy. Paired samples of breast cancer FNAs collected pre-operatively and post-operatively were compared with tissue samples obtained at the time of surgical resection. Western blots were probed for p53 using the antibodies DO12 and DO1, and for levels of downstream proteins p21/WAF1 and p27. The protein extracted by FNA was sufficient for up to 5 Western blot studies. p53 expression and phosphorylation did not differ significantly pre- and post-operatively, indicating that intra-operative manipulation does not affect p53 expression or downstream activation in breast cancer. However, expression of p53, p21 and p27 varied between individual patients suggesting a range of p53 pathway activation in breast cancer. Immunohistochemistry confirmed that the cancer cells accounted for the protein expression detected on Western blots. FNA yields adequate protein for Western blotting studies and could be used as a method to monitor p53 activity in vivo before and during anti-cancer treatment possibly providing early evidence of tumour response to therapy.

Biopsy, Needle↗
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↗
PubMed2001

Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.

Amino Acid Substitution↗
PubMed2000

Strategies for manipulating the p53 pathway in the treatment of human cancer.

Human cancer progression is driven in part by the mutation of oncogenes and tumour-suppressor genes which, under selective environmental pressures, give rise to evolving populations of biochemically altered cells with enhanced tumorigenic and metastatic potential. Given that human cancers are biologically and pathologically quite distinct, it has been quite surprising that a common event, perturbation of the p53 pathway, occurs in most if not all types of human cancers. The central role of p53 as a tumour-suppressor protein has fuelled interest in defining its mechanism of function and regulation, determining how its inactivation facilitates cancer progression, and exploring the possibility of restoring p53 function for therapeutic benefit. This review will highlight the key biochemical properties of p53 protein that affect its tumour-suppressor function and the experimental strategies that have been developed for the re-activation of the p53 pathway in cancers.

Animals↗
PubMed2000

Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription.

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

Antibodies, Monoclonal↗
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↗
PubMed2000

The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A.

Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B(1)-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B(1)-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.

Amino Acid Motifs↗
PubMed2000

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA.

Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53-DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53-DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53-mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA.

Blotting, Western↗
PubMed2000

P53 and breast cancer.

p53 mutation is the most common genetic abnormality found so far in human cancer, and in breast cancer p53 mutation/alteration is seen in up to 50% of primary carcinomas. Together with the increasing knowledge of the characteristics and understanding of the role of p53 over the last two decades, attention in recent years has been focused on how this knowledge can be used in clinical settings for patient care and management in terms of analyzing p53 as a potential marker for studying the relationship between p53 expression and tumour development, progression and outcome; and designing alternative treatment strategies specifically aimed at restoring normal p53 function.

Journal Article↗
PubMed1999

Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein.

In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.

Antibodies, Monoclonal↗