Steven M. Picksley

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li–Fraumeni and Li–Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with ≈amp;60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

p53 and Rb: their cellular roles

Within the past year considerable new insights have been gained into the roles the p53 and retinoblastoma tumour suppressors play in determining the fate of cells through their regulation of cell cycle progression, apoptosis and gene expression. Key advances have been achieved in the identification and characterization of functional domains and through functional knockout studies.

Human hair follicles contain two forms of ATP-sensitive potassium channels, only one of which is sensitive to minoxidil

Hair disorders cause psychological dis tress but are generally poorly controlled;more effective treatments are required. Despite the long‐standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP‐sensitive potassium (KATP) channels. To investi gate whether KATP channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tol butamide, a KATP channel blocker, opposed each oth ers effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase‐polymerase chain reaction identi fied KATP channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore‐forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were exam ined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhib ited SUR2B and Kir6.1. Immunohistochemistry demon strated similar protein distributions. Thus, human fol licles respond biologically to KATP channel regulators in culture and express genes and proteins for two KATPchannels, Kir6.2/SUR1 and Kir6.1/SUR2B;minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain KATPchannels that can respond to minoxidil and that tolbu tamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.— Shorter, K., Farjo, N. P., Picksley, S. M., Randall, V. A. Human hair follicles contain two forms of ATP‐sensitive potassium channels, only one of which is sensitive to minoxidil. FASEB J. 22, 1725–1736 (2008)

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li – Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

Increased epidermal functioning wild-type p53 expression in vitiligo

Abstract:  Despite the lack of protective melanin and increased oxidative stress due to mM concentrations of epidermal H2O2 in vitiligo, there is no significantly increased risk for chronic actinic damage and non‐melanoma skin cancer. Therefore the question arises, which protective mechanisms could be involved in the skin of these patients preventing the initiation of these cancers. Recently an overexpression of p53 has been shown in vitiligo. Unfortunately there was no further characterization of this elevated p53. Employing a functional colour yeast assay, the study presented herein demonstrates for the first time the overexpression of a functioning wild‐type p53 protein in both depigmented and ‘normal’ pigmented epidermis of patients with vitiligo compared with healthy controls. Surprisingly long‐term narrowband UVB (311 nm) treatment does not alter this expression. Moreover, MDM‐2, PCNA and p21 protein expression remain unchanged compared with healthy controls. This increased epidermal p53 in vitiligo coincides with decreased thioredoxin reductase (TR) protein levels in both depigmented and pigmented skin whereas mRNA expression is unaffected. Because TR is one transcriptional target of p53, these results support a wild‐type functionality, which was further supported by the specific p53 FASAY yeast test. To our knowledge this is the first example of persistent elevated functioning wild‐type p53 in humans. Based on our results we hypothesize that the low incidence for actinic damage, basal cell and squamous cell carcinoma as documented in vitiligo could well reside in a protective function of up‐regulated wild‐type p53.

The prostamide-related glaucoma therapy, bimatoprost, offers a novel approach for treating scalp alopecias

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side‐effects of prostamide F2α‐related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non‐eyelash follicles could respond to bimatoprost, a prostamide F2α analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor‐mediated mechanism within follicles; RT‐PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost‐sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low‐risk therapeutic approach for scalp alopecias.—Khidhir, K. G., Woodward, D. F., Farjo, N. P., Farjo, B. K., Tang, E. S., Wang, J. W., Picksley, S. M., and Randall, V. A. The prostamide‐related glaucoma therapy, bimatoprost, offers a novel approach for treating scalp alopecias. FASEB J. 27, 557–567 (2013). www.fasebj.org

The P53-MDM2 Interaction in a Cancer-Prone Family, and the Identification of a Novel Therapeutic Target

One approach to developing novel anti-cancer agents is to identify and characterise targets that directly regulate cell growth and are dysfunctional in the disease state. One such target is the interaction between the p53 tumour suppressor and the oncogene product of the murine double minute gene, MDM2. MDM2 is known to bind wild-type p53 and block the transcriptional activation of p53-dependent genes. We have previously described a cancer-prone family with elevated levels of wild-type p53, and now show that MDM2 is also over-expressed in the proband from this family. Interestingly, the overexpression of MDM2 is independent of other p53-regulated genes such as p21WAF1. The present work and a review of recent insights into the p53-MDM2 interaction, and p53 transcriptional activity, identify a new target site for the rational development of novel anti-cancer agents.

Analysis of the K-ras and p53 pathways in X-ray-induced lung tumors in the rat.

The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to limited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT --> GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. Thus the increased level of p53 protein in one SCC may stem from stabilization by the mdm2 gene product. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. Overall homology between the rat and human cip1 genes was 74%. Homology between the rat and mouse genes was 90%. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat.

Proteins from Yeast and Human Cells Specific for Model Holliday Junctions in DNA

Genetic recombination involves the exchange of genetic material between chromosomes to produce new assortments of alleles. As such, it affects one of the most fundamental and important components of heredity, the genome itself. Genetic rearrangements can be favourable or unfavourable, and certain forms of cancer have been linked to gene translocations. To understand the molecular basis of recombination, we have directed our efforts to try to determine how simple organisms recombine their DNA. In bacteria and lower eukaryotes, the enzymes involved in genetic recombination also play a role in the repair of DNA following irradiation or chemical damage. This overlap between recombination and repair is indicative of a need for recombinational repair, a process which ensures that the integrity of the chromosomal material is maintained.