Björn Sjöström

Differential expression of p63 isoforms in normal tissues and neoplastic cells

The p63 gene encodes at least six different proteins with homology to the tumour suppressor protein p53 and the related p53 family member p73. So far, there have been limited data concerning the expression patterns of individual p63 proteins, due to a lack of reagents that distinguish between the different isoforms. Three antibodies have been produced specifically directed against the two N‐terminal isoforms (TAp63 and ΔNp63) and the C‐terminal region of the p63α proteins. TAp63 proteins are located suprabasally in stratified epithelia compared with the N‐terminal truncated forms, which are more abundantly expressed in the basal cell layer, indicating a switch in expression of p63 isoforms during normal cellular differentiation. Analysis of squamous cell carcinomas shows ΔNp63α to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. ΔNp63 protein expression is also restricted to basal cells in breast and prostate, whilst TAp63 isoforms are more widely expressed in these tissues as well as in tumours at these sites. TAp63, but not ΔNp63 or p63α, is detected in normal colon and in colon carcinoma. TAp63 proteins are also expressed in the nuclei of a sub‐population of lymphoid cells and in most malignant lymphomas, whereas ΔNp63 proteins are not expressed. Taken together, a hitherto unrecognized regulation of p63 isoform expression in vivo has been uncovered, with different p63 proteins expressed during differentiation and in different cell types. The data indicate roles for specific p63 isoforms not only in maintaining epithelial stem cell populations, but also in cellular differentiation and neoplasia. Copyright

The Clinical Value of Image Cytometry DNA Analysis in Distinguishing Branchial Cleft Cysts From Cystic Metastases of Head and Neck Cancer

Objectives/Hypothesis A branchial cleft cyst presents as a lump in the neck that, generally, is easily cured by surgical excision. The preoperative diagnosis is based on clinical examination and, especially in the Scandinavian countries, fine‐needle aspiration cytology. However, at times, the histopathological analysis of the excised cyst reveals a cystic metastasis of squamous cell carcinoma of the head and neck. If adequate diagnosis could be obtained preoperatively, patients would most likely fare better. The study was performed to investigate whether the diagnostic accuracy for these lesions could be improved preoperatively by image cytometry DNA analysis of the fine‐needle aspiration cytology specimen.

Function and importance of p63 in normal oral mucosa and squamous cell carcinoma of the head and neck.

BACKGROUND/AIMS Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common malignancy worldwide with a 5-year survival that has not improved over the last 20-25 years. Factors of prognostic significance for this tumour type include the presence of regional lymph node metastasis and amplification of chromosome 3q21-29, where the p63 gene is located. This gene encodes 6 proteins and is crucial for formation of the oral mucosa, teeth, salivary glands and skin. Each of the 6 different p63 proteins has different characteristics and functions, where some resemble the tumour suppressor protein p53, whilst others have functions that oppose p53. METHODS To understand the function and importance of p63 in oral mucosa and tumour development we have studied protein as well as mRNA expression in normal oral mucosa and tumours. RESULTS/CONCLUSION Expression of p63 proteins differs between the cell layers in normal oral mucosa, and primary HNSCC has a high expression level of p63 isoforms normally expressed in basal cells. Data suggest that p63 expression in HNSCC influences tumour cell differentiation.

Cytoskeletal Identification of Intermediate Filaments in the Inner Ear of the Jerker Mouse Mutant

The expression of the five main groups of intermediate filaments (IF) and their subgroups, especially cytokeratins, was analysed in cryosections of the labyrinth of the jerker mouse mutant and compared with normal CBA/CBA controls. Fourteen different well characterized monoclonal antibodies were used. In principle the same pattern of IF was found in the two mouse strains. IF were not found in hair cells of the mouse inner ear, which may reflect a disparity as compared with human fetal hair cells. Chain-specific (epitope-specific) differences were visualized in cytokeratin no. 8 with regard to cells in epithelia involved in the homeostasis of inner ear fluids. Differences in immuno-staining occurred between adjacent cells in the same epithelium, for instance in the semicircular canals.

Cochlear structure and function in a recessive type of genetically induced inner ear degeneration

An age-related consecutive morphological analysis of the cochlea has been performed in homozygote (je/je) and heterozygote (je/+) jerker mouse mutants. A difference in the time of onset of hair cell pathology was evident between the two, but, when taking place, it showed a similar morphological type of degeneration. The cuticular plate and the stereocilia are particularly vulnerable structures and are the primary sites of damage. The suprastructures on both outer and inner hair cells disintegrate at the same time, irrespective of the level along the basilar membrane. During aging, the je/+ animals showed a progressive age-related impairment of auditory brainstem response thresholds which was correlated semiquantitatively to hair cell pathology.

TRAF4 is potently induced by TAp63 isoforms and localised according to differentiation in SCCHN

p63, a member of the p53 family, is overexpressed in squamous cell carcinoma of the head and neck (SCCHN) and some other tumors of epithelial origin. As a transcription factor, p63 can bind to p53-type response elements and there is some overlap between p53 family transcriptional targets. Tumor necrosis factor receptor associated factor 4 (TRAF4) is a p53 regulated gene which is overexpressed in many human carcinomas. We investigated the involvement of p63 in regulation of TRAF4 and the expression of the TRAF4 protein in SCCHN. Disrupting endogenous p63 expression resulted in downregulation of TRAF4 mRNA and protein in an SCCHN cell line. Endogenous p63 bound to the TRAF4 promoter in vivo and reporter assays showed that p63, p73 and p53 can all transactivate TRAF4, with TAp63 isoforms being the most potent activators. The level of TRAF4 activation by TAp63 was two-fold higher than by p53, and TRAF4 was 10-fold more responsive to TAp63 than another p63-target, IGFBP3. Nuclear expression of TRAF4 was seen in normal oral epithelium and highly/moderately differentiated SCCHN, whereas cytoplasmic expression of TRAF4 was seen in poorly differentiated SCCHN. These results indicate that TRAF4 is a common target of p53 family members and that localization of TRAF4 is associated with differentiation of SCCHN cells.

Variability In Genetically Induced Age-related Impairment of Auditory Brainstem Response Thresholds

In genetically induced hearing impairment or deafness, the recessive mode of inheritance is by far the most common, but it has seldom been feasible to document audiological aberrations. In this study, auditory brainstem response (ABR) thresholds were analysed in groups of 10 heterozygote jerker (je/+) mice each, at ages of 3, 6 and 9 months in the frequency range 2-31.5 kHz. The animals were thereafter regularly tested up to a final age of 12 months. Forty age-related CBA/CBA mice were used as controls. The individual ABR thresholds in 3-, 6-, 9- and 12-month-old CBA/CBA mice were very similar, as also were those in 3- and 6-month-old je/+ mice (mean values within the normal range). In contrast, at 9 months--and even more so at 12 months--all je/+ animals showed large individual variations, with ABR thresholds varying randomly from near-normal to profound impairment. The wide individual spread of ABR thresholds in old je/+ animals was interpreted as reflecting the expression of a random influence of recessive jerker vis-à-vis the wild type of gene.

Cell kinetic changes in human squamous cell carcinomas during radiotherapy studied using the in vivo administration of two halogenated pyrimidines

The aim of this study was to investigate cell cycle changes during radiation treatment and establish whether treatment intervention could be considered if these changes helped to predict outcome. 33 patients with head and neck cancer were administered iododeoxyuridine (IdUrd) prior to treatment and a second administration of bromodeoxyuridine (BrdUrd) prior to the fifth fraction of 2 Gy. Biopsies were taken several hours after each injection and flow cytometry was used to calculate changes in the cellular kinetics and cell cycle delay in vivo. The kinetic response of the tumour cells was variable; some showed an increase in proliferation during the first week of treatment, whilst the majority showed an inhibition of proliferation. Reduction in the labelling index (LI) and the pretreatment DNA ploidy status and not delays in G2 were the only parameters to correlate with clinical outcome. A lack of reduction in the LI after 1 week of radiotherapy and DNA aneuploidy predicted a group of patients where radiotherapy failed. This information could be helpful in planning future treatment interventions.

Cochlear Synaptic Development and Morphology in a Genetically Induced Type of Progressive Hair Cell Degeneration

In mice with genetically induced inner ear abnormalities it is conceivable that in the morphogenetic types and in mutants with the spotting kind of pigmentary anomaly, the genes act through the developing nervous system. It has been suggested that in degenerative (neuroepithelial) mutants the influence of the gene is also reflected in the inner ear through the agency of the nervous system. The jerker mouse belongs to the neuroepithelial type of mutants which in homozygotes results in early postnatal degeneration of the sensory epithelium of the inner ear, initially confined to the cuticular plate and the stereocilia. In spite of well-advanced hair cell degeneration, these mutants developed morphologically normal afferent and efferent nerve terminals at cochlear hair cells.