Peter A. Hall

The p53 pathway

Abnormalities of the p53 tumour suppressor gene are among the most frequent molecular events in human and animal neoplasia. Moreover, p53 is one of the most studied proteins in the whole of contemporary biology, with more than 12500 papers so far written! In this review the choice has been deliberately made not to be fully comprehensive in the coverage of the huge p53 literature. Rather attention is focused on a small number of recent developments which are reviewed in the context of modern models of p53 function. Progress in the analysis of signalling to p53 including phosphorylation cascades, and interactions with proteins such as mdm2 and ARF are highlighted. The plethora of protein–protein interactions is discussed, as are the strategies for defining downstream targets of p53. Finally, the emerging biology of p53 homologues is considered. The need for bridging the gap between reductionist, biochemical and biophysical studies and biological and genetic analysis is emphasized. Only this will provide the needed framework for utilizing the information in clinical care. Copyright

Detection of the Ki-67 antigen in fixed and wax-embedded sections with the monoclonal antibody MIB1

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki‐67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki‐67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki‐67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki‐67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nulcear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo—ultraviolet irradiated human skin. MIB1 staining correlated with semi‐conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki‐67. Thus, MIB1 represents a new anti‐Ki‐67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.

The prohibitin family of mitochondrial proteins regulate replicative lifespan

Cellular senescence is determined by multiple factors, including the genetic regulation of metabolism and responses to endogenous and exogenous stresses [1-4]. Recent studies implicate a limited number of gene products in elongating lifespan in yeast and Caenorhabditis elegans [2-4]; these include the C, elegans gene cik-1, a central regulator of metabolism [5], and yeast RAS2, which controls the response to ultraviolet irradiation and other stresses [3]. Another gene postulated to effect senescence is PHB1, the yeast homologue of prohibitin [3], a rodent gene initially identified as a potential regulator of growth arrest and tumour suppressor [6-8]. Highly conserved prohibitin homologues have been identified in mammals [9], Drosophila [10], C. elegans [9], plants [11] and yeast. A second mammalian gene, encoding BAP37, a protein with sequence similarity to prohibitin, is thought to be involved in lymphocyte function [9]. Here, we show that the nuclear-encoded mammalian prohibitin and BAP37 proteins are present in mitochondria, are co-expressed, and interact physically with each other. Deletion of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, results in a decreased replicative lifespan and a defect in mitochondrial membrane potential. Our observations highlight the relationship between the metabolic efficiency of cells and the ageing process, and provide evidence for its evolutionary conservation.

Characterization of the expression pattern of p63α and δnp63α in benign and malignant oral epithelial lesions

The p53 homologue p63 is essential for ectodermal differentiation, such that p63−/− mice lack all squamous epithelia and teeth. The p63 gene expresses at least 6 different transcripts, but information regarding the expression, regulation and function of the different isoforms has remained sparse, due to the lack of adequate reagents directed specifically against the individual proteins. Here we characterize the expression of p63α/ΔNp63α in benign and malignant lesions of the oral epithelium, using a specific antibody raised against a peptide derived from the C‐terminus of p63α, which does not cross‐react with p53 or the other p53 homologue, p73. By immunohistochemical analysis, we show that these p63 isoforms are expressed in the nucleus of many cells. In normal and benign lesions, p63α/ΔNp63α‐expressing cells are mainly found suprabasally, whereas p53‐expressing cells are restricted to the basal‐cell layer. By RT‐PCR, we show that ΔNp63α is the predominant isoform in cell lines from squamous‐cell carcinomas of the head and neck, confirming our immunochemical observations. Our data are consistent with studies suggesting a role for p63 in the transit‐amplifying population of epidermal cells. Over‐expression of p63α, and in particular the ΔN form, was frequently seen in carcinomas. Taken together with previous analyses of p63 expression, our data suggest distinct roles for different p63 isoforms in the regulation of growth and/or differentiation of epithelial cells. Moreover, our data are compatible with the notion that p63 can act to promote neoplastic growth in the oral epithelium. Int. J. Cancer 87:368–372, 2000.

The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle

Although widely used as a marker of cell proliferation, the biochemical properties and function of the Ki67 antigen remain poorly understood. Recent data indicate that it can interact with RNA, DNA, and a number of cellular proteins including elements of the ubiquitin proteolytic pathway and a novel kinase. The evidence for its expression only in cycling cells is extensive and it is not regulated by stress, apoptosis or DNA damage. It was reasoned that a detailed characterization of the localization of pKi67 and analysis of its spatial relationship to other nucleolar proteins may provide insights into its function. Using high‐resolution laser scanning confocal microscopy with double and triple labelling, pKi67 expression in MCF7 cells has been defined in relation to the distribution of nucleolin, fibrillarin, p130 (human Nopp 140 homologue), p120 (Nol 1), RH‐II/Gu helicase, and topoisomerase II β. All of these molecules are perichromosomal during mitosis and all but fibrillarin and p130 show extra‐nucleolar distribution in early G1. The majority of p120 (Nol 1) and RH‐II/Gu helicase co‐localize in the diffuse fibrillar centre (DFC) of nucleoli, while there is only partial overlap with nucleolin and fibrillarin. There is no co‐localization between p130 and pKi67. These data refine current understanding of the distribution of pKi67 and its physical relationship with functional domains of the nucleolus and place pKi67 in a zone of the DFC associated with late rRNA processing. Taken together with recent biochemical data, these observations allow the proposal of a model of pKi67 function in which it acts as an ‘efficiency factor’ in ribosome biogenesis during the heavy metabolic demands placed on a cell during the cell division cycle. Copyright

Expression of the 'dead box' RNA helicase p68 is developmentally and growth regulated and correlates with organ differentiation/maturation in the fetus

The human DEAD box protein p68 is an established RNA‐dependent ATPase and RNA helicase. p68 has been highly conserved in evolution and appears to be essential for normal growth, suggesting that this protein plays an important role in the cell. Although the biochemical activities of p68 are fairly well characterized, little is known about its biological function. This report shows that p68 is detectable in quiescent cell lines, but its expression is induced by serum, suggesting that this protein may play a role in cell growth. It is also shown that both p68 mRNA and protein are differentially expressed in adult tissues; in this case, however, the levels do not always correlate with proliferation status, suggesting that the regulation of expression in the animal may be different from that in cell lines. Finally, it is shown that p68 expression is developmentally regulated and appears to correlate with organ differentiation/maturation. These findings suggest that p68 expression may not simply reflect proliferation/differentiation status and that it appears to be regulated in a more complex way.

APC EXPRESSION IN NORMAL HUMAN TISSUES

The tumour suppressor gene APC codes for a 2843‐amino acid protein whose precise functions are still poorly understood. This paper describes the development of two new antisera to APC (to amino‐ and carboxy‐terminal epitopes) which permit localization of the protein by immunohistochemistry in archival paraffin sections. The protein is expressed in a wide variety of normal epithelial tissues. Its distribution frequently coincides with the location of post‐replicative cells within tissues. Staining patterns demonstrate that the APC protein, although often diffusely cytoplasmic in distribution, may also accumulate in the apical and immediately subapical regions, or along the lateral margins of certain cells. These results indicate that APC is significant in many tissues in addition to the colorectal epithelium. They are compatible with a function related to signalling at the adherens junction and possibly with other more complex roles in cells committed to terminal differentiation.

Tumour suppressors: A developing role for p53?

An increasing body of evidence indicates that p53, the product of a tumour suppressor gene, has a role in development - could this developmental role have provided the primary driving force in the evolution of a protein best known as a stress-response integrator?

Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta and is an absolute requirement for cellular proliferation. Specialized-type Barretts columnar-lined esophagus (CLE) is associated with adenocarcinomatous change. In the present study, the cellular proliferation of three histological types of CLE was assessed by semiquantitative evaluation of PCNA immunolocalization in 93 biopsy specimens from 45 patients using the murine monoclonal PC10. Statistical comparison was performed by the Mann-Whitney U test. Luminal surface cell labeling was uncommon in all histological types other than specialized CLE where 25 of the 43 biopsy specimens had at least occasional luminal surface cell labeling. Mean crypt labeling score of 4.06 for specialized type exceeded that for junctional (mean, 3.12; P < 0.001) and fundic types (mean, 1.6; P < 0.001). Gland cell PCNA staining scores for specialized-type CLE (mean, 3.18) exceeded that of junctional (mean, 1.97; P < 0.001) and fundic (mean, 1.04; P < 0.001). Summated PCNA scores for specialized-type, mean of 8.29, exceeded junctional mean score of 5.45 (P < 0.001) and fundic mean score of 2.76 (P < 0.001). PCNA immunolocalization reveals a high proportion of cells in cycle in the specialized-type CLE and expansion of the proliferative compartment, which may explain the association of specialized-type CLE with malignancy.

Transcriptional activation of tyrosinase and TRP‐1 by p53 links UV irradiation to the protective tanning response

We are exposed constantly to potentially harmful compounds and radiations. Complex adaptive protective responses have evolved to prevent such agents causing cellular damage, including potentially oncogenic mutation. The p53 tumour suppressor appears to have a role in co‐ordinating such responses: it is activated by diverse insults and it acts as a transcriptional regulator of downstream genes that facilitate cellular adaptation. Ultraviolet (UV) light is a particularly potent inducer of p53 expression. In addition, UV light induces the production of melanin as a protection against further irradiation‐induced damage. This study shows that the promoters of the genes coding for the enzymes crucial in melanin biosynthesis, namely tyrosinase and tyrosinase‐related protein‐1 (TRP‐1), are activated by wild‐type p53. Both promoters have p53‐responsive elements and are activated in vivo in a dose‐dependent manner by wild‐type p53, as well as by the p53 homologues p73α and p63α. Copyright