Michael Slater

Skin cancer prevention: A possible role of 1,25dihydroxyvitamin D3 and its analogs

We previously reported that the natural hormone 1,25dihydroxyvitamin D3 (1,25(OH)(2)D(3)) protects human skin cells from ultraviolet radiation (UVR)-induced apoptosis. UVR-induced pre-mutagenic cyclobutane pyrimidine dimers are diminished in number from 0.5h after cessation of UVR in all skin cell types, by treatment with three different Vitamin D compounds: by 1,25(OH)(2)D(3), by the rapid acting, low calcemic analog, 1alpha,25(OH)(2)lumisterol(3) (JN) and by the low calcemic but transcriptionally active hybrid analog 1alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3 QW-1624F2-2 (QW), which may explain the enhanced cell survival. The rapid response antagonist analog 1beta,25(OH)(2)D(3) (HL) abolished the photoprotective effects of 1,25(OH)(2)D(3) whilst a genomic antagonist, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647), had no effect. UVR increased p53 expression in human skin cells, whilst concurrent treatment with 1,25(OH)(2)D(3) further enhanced this effect several fold, at 3 and 6h after UVR. Combined with previously reported lower nitrite levels with 1,25(OH)(2)D(3), this increased p53 expression may favor DNA repair over apoptosis. We now report that topical application of 1,25(OH)(2)D(3) or QW also suppressed solar simulated UV (SSUVR-induced pyrimidine dimers in the epidermis of irradiated hairless Skh:HR1 mice, measured 24h after irradiation. Furthermore, UVR-induced immunosuppression in the mice was markedly reduced by topical application of either 1,25(OH)(2)D(3) or QW. These preliminary results show, for the first time, a protective effect of Vitamin D compounds against DNA photodamage in vivo.

Early prostate cancer detected using expression of non-functional cytolytic P2X7 receptors

Aims:  To detect early prostate cancer reliably by monitoring the expression of non‐functional P2X7 cytolytic purinergic receptors.

Purinergic receptor distribution in endothelial cells in blood vessels: a basis for selection of coronary artery grafts.

Expression levels of the purinergic P2X receptor subunits (P2X(1) to P2X(7)) and P2Y(2) were examined in the endothelial cell layer of internal mammary artery (Ann. Thorac. Surg. 54 (1992) 652), radial artery (Ann. Thorac. Surg. 16 (1973) 111) and saphenous vein (Ann. Thorac. Surg. 20 (1975) 628) samples obtained at surgery for coronary artery bypass grafts using immunohistochemistry and confocal microscopy. Similar levels of P2X(1), P2X(2), P2X(3), P2X(7) and P2Y(2) were found in the endothelial cells in all vessels examined while the levels of P2X(5) and P2X(6) were uniformly lower. A clear difference was measured in P2X(4) expression between arteries and veins. Both radial and internal mammary arteries exhibited very low levels of P2X(4) whereas the level in the saphenous vein was 14.6 fold higher (P<0.0001), approaching that of the major receptor subtypes. These data showing strong expression of P2X(4) in veins have implications for the choice of vessels used in coronary artery bypass grafts given that P2X(4) is involved in calcium influx into endothelial cells, modulates blood vessel contractility and is up-regulated in situations involving intima proliferation suggesting vein grafts are more susceptible to developing atherosclerosis.

An Akt-dependent Increase in Canonical Wnt Signaling and a Decrease in Sclerostin Protein Levels Are Involved in Strontium Ranelate-induced Osteogenic Effects in Human Osteoblasts

Sclerostin is an important regulator of bone homeostasis and canonical Wnt signaling is a key regulator of osteogenesis. Strontium ranelate is a treatment for osteoporosis that has been shown to reduce fracture risk, in part, by increasing bone formation. Here we show that exposure of human osteoblasts in primary culture to strontium increased mineralization and decreased the expression of sclerostin, an osteocyte-specific secreted protein that acts as a negative regulator of bone formation by inhibiting canonical Wnt signaling. Strontium also activated, in an apparently separate process, an Akt-dependent signaling cascade via the calcium-sensing receptor that promoted the nuclear translocation of β-catenin. We propose that two discrete pathways linked to canonical Wnt signaling contribute to strontium-induced osteogenic effects in osteoblasts.

Differentiation between cancerous and normal hyperplastic lobules in breast lesions.

Determining the risk that a particular area of hyperplastic breast tissue will progress to cancer is difficult and is currently expressed only as a general risk factor within the population. Using an antibody against the apoptotic purinergic receptor P2X7, we examined 40 cases each of the following histological categories: normal, moderate, florid and atypical hyperplasia, lobular carcinoma in situ, ductal carcinoma in situ, invasive lobular and invasive ductal carcinoma. These were previously diagnosed by H&E and supplied by clinical laboratories as tissue sections. Normal and mildly hyperplastic epithelium was devoid of the cytolytic P2X7 receptors whereas all epithelial cells in all cases of in situ or invasive lobular or ductal carcinoma labelled intensely. The lobular and ductal in situ cases labelled intracellularly while the invasive epithelial cancer cells showed intense cell surface label indicating an attempt was being made to induce apoptosis. All these receptors however are non-functional and thus unable to induce apoptosis. Approximately 10% of all hyperplastic lobules examined in the biopsied tissue, regardless of H&E classification, labelled for P2X7, which is suggestive of early metabolic cancerous change. The acini within lobules were either completely labelled with P2X7 or were completely devoid of the receptor. A potential advantage of this method lies in identifying early cancerous change in hyperplastic lobules and in establishing the true extent of cancerous spread in infiltrating lesions, thus facilitating the task of reporting clear surgical margins.

Endometrial cell death during early pregnancy in the rat.

In a study of early pregnancy in the rat, a high proportion of morphologically apoptotic, TUNEL and P2X7 positive cells were found to be present in the luminal epithelium and stroma prior to implantation. At the time of implantation on Day 6, apoptosis as measured by these indicators was reduced up to 4-fold in the non-implantation uterine epithelium but was markedly increased adjacent to the implanting blastocyst. It is proposed that apoptotic cell death is an important regulatory factor involved in uterine remodelling prior to and during implantation.

Expression of the apoptotic calcium channel P2X7 in the glandular epithelium.

In the current study, expression of the apoptotic calcium channel receptor P2X7 and prostate-specific antigen (PSA) levels were studied in biopsy cores from 174 patients as well as 20 radical prostatectomy cases. In clinical biopsies, we have previously demonstrated that P2X1 and P2X2 calcium channel receptors are absent from normal prostate epithelium that does not progress to prostate cancer within 5 years. In cases that did progress to prostate cancer however, P2X1 and P2X2 labeling was observed in a stage-specific manner first in the nucleus, then the cytoplasm and finally on the apical epithelium, as prostate cancer developed. These markers were present up to 5 years before cancer was detectable by the usual morphological criteria (Gleason grading) as determined by H&E staining. In the current study, the apoptotic calcium channel receptor P2X7 yielded similar results to that of P2X1 and P2X2. Using radical prostatectomy tissue sections as well as biopsies, these changes in calcium channel metabolism were noted throughout the prostate, indicating a field effect. This finding suggests that the presence of a prostate tumor could be detected without the need for direct sampling of tumor tissue, leading to detection of false negative cases missed by H&E stain. The reliability of PSA levels as a prognostic indicator has been questioned in recent years. In the current study, PSA levels were correlated with the P2X7 labeling results. All patients who exhibited no P2X7 labeling had a prostatic serum antigen (PSA) level of <2. Patients who exhibited stage-specific P2X7 expression, and who later developed obvious prostate cancer as diagnosed by H&E stain, all had a PSA > 2. This finding suggests that increasing PSA may be an accurate indicator of cancer development.

Distributional changes of purinergic receptor subtypes (P2X 1-7) in uterine epithelial cells during early pregnancy.

Expression of each of the purinergic receptor subtypes (P2X1–7) was studied by immunohistochemical localization in the apical, lateral and basal plasma membranes of rat uterine epithelial cells during early pregnancy to the time of implantation on Day 6. Labelling for each P2X subtype was seen in the apical, lateral and basal compartments on Days 1 and 3, except for P2X2 which was only observed in the basement membrane. The P2X5 signal was similar in temporal and spatial expression to the other subtypes, but with a greatly reduced intensity. At the time of implantation on Day 6, this pattern altered dramatically. Apical expression markedly increased for most subtypes while the lateral and basal signals were markedly reduced. The exceptions to this pattern were P2X2, which displayed both a strong basal and apical label, and P2X4 which became de-expressed in all areas. We propose that the changing spatial and temporal expression of the P2X receptors is a significant factor in the regulation of events during early pregnancy. They are expressed in the same location as remodelling, apoptosis, and protein activation events prior to implantation on Day 6. These observations suggest an up-regulation of calcium-mediated events, including cytoskeletal alterations, a decrease in luminal pH and transmembrane molecule activation.

Differentiating keratoacanthoma from squamous cell carcinoma by the use of apoptotic and cell adhesion markers

Aims : Keratoacanthomas (KA) are well‐differentiated squamoproliferative skin lesions that grow rapidly and regress spontaneously. In contrast, squamous cell carcinomas (SCC) can have variable differentiation, inexorably progress and on occasion metastasize. Distinguishing between KA and SCC using haematoxylin and eosin‐stained sections from an initial biopsy can often be difficult. There is also some debate as to whether KA is simply a variety of well‐differentiated SCC or a distinct entity.

Markers for the development of early prostate cancer

Biochemical and genetic changes precede histologically identifiable changes accompanying cell transformation often by months or years. De‐expression of the extracellular matrix adhesive glycoprotein tenascin and the cell‐to‐cell adherent protein E‐cadherin have been suggested as markers of early neoplastic change in prostate epithelial cells. Previous studies have been inconclusive, probably due to epitope masking. This study examined 2378 biopsy cores from 289 prostates using a heat antigen retrieval protocol at low pH to improve the accuracy of detection. Tenascin and E‐cadherin de‐expression was correlated with purinergic receptor and telomerase‐associated protein labelling, as well as prostate‐specific antigen (PSA) levels and Gleason scores. E‐cadherin was a poor marker, as it was expressed in all lesions except carcinomas of the highest Gleason score. Tenascin was maximally expressed in the extracellular matrix and acinar basement membrane in normal and prostatic intraepithelial neoplasia tissue. In prostate cancer tissue, tenascin expression did not correlate with Gleason score but was significantly de‐expressed as purinergic receptor and telomerase‐associated protein expression increased. Marked changes in tenascin, telomerase‐associated protein, and purinergic receptor expression were apparent before any histological abnormalities were visible by haematoxylin and eosin (H&E) stain, making these potential markers for early and developing prostate cancer. Moreover, the potential increased accuracy of diagnosis of underlying prostate cancer using purinergic receptor translocation (PRT) assessment suggests that PSA levels may be more accurate than has generally been supposed when apparent false negatives arising from H&E‐based diagnoses are correctly categorized. Copyright