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.

Vitamin D is produced in skin by UVB radiation (290-320 nm) acting on 7-dehydrocholesterol. The hypotheses that the active vitamin D hormone, 1,25 dihydroxyvitamin D-3 (1,25(OH)(2)D-3), would increase the survival of skin cells after UV irradiation and that surviving cells after 1,25(OH)(2)D-3 treatment would have no increase in DNA damage were tested. The survival of keratinocytes post-UVR was significantly greater after treatment with 1,25(OH)(2)D-3 compared to vehicle (P < 0.01). Significant reductions in thymine dimers (TDs) in surviving keratinocytes after UVR were noted in the presence of 1,25(OH)(2)D-3 (P < 0.001). Nuclear p53 protein expression increased after UVR and was significantly higher in keratinocytes treated with 1,25(OH)(2)D-3 (P < 0.01), whereas NO products were significantly reduced (P < 0.05). Both the increase in nuclear accumulation of p53 protein and reduced formation of nitric oxide products may contribute to the reduction in TDs seen with 1,25(OH)(2)D-3 after UVR. Reductions in numbers of sunburn cells (P < 0.01) and in TDs (P < 0.05) were observed 24 hours after UVR in skin sections from Skh:hr1 mice treated with 1,25(OH)(2)D-3. These results are consistent with the proposal that the vitamin D system in skin may be part of an intrinsic protective mechanism against UV damage.

In this retrospective study on banked tissue, we found that alpha-actinin and talin were completely de-expressed in both endometriosis and endometrioid carcinoma tissue. Some patchy, depolarized labeling for ezrin was noted in the endometrioid carcinoma but not in endometriosis. The loss of these proteins in both endometriosis and endometrioid carcinoma tissue indicates a significant change in the integrity of these tissues compared with normal and the possibility that individual cells may break away from the parent histology due to loss of cell adhesion. It also indicates a similarity between endometrioid cancer and endometriosis with respect to epithelial cell function and adhesion.

In this retrospective and quantitated study on banked tissue we found that, compared to normal uterine epithelial cells, growth hormone (GH) is increased 3.4-fold in endometriosis and 3.8-fold in endometrial adenocarcinoma. Similarly, interleukin-6 (IL-6) is increased 2.4-fold in endometriosis and 4.4-fold in endometrial adenocarcinoma. These proteins appear to be involved in the progression of both these conditions. GH is particularly interesting in this context since it is known to not only promote cellular proliferation but also reduces cell–cell adhesion, thus allowing individual cells to break away from their parent architecture. Our results suggest that both IL-6 and GH may play a role in the progression of both endometriosis and endometrial carcinoma.

In studies involving mice in which doses of UVA (320-400 nm) and UVB (290-320 nm) radiation were administered alone or combined sequentially, we observed a protective effect of UVA against UVB-induced erythema/edema and systemic suppression of contact hypersensitivity. The UVA immunoprotection was mediated by the induction of the stress enzyme heme oxygenase-1 (HO-1) in the skin, protection of the cutaneous Th1 cytokines interferon-gamma (IFN-gamma) and IL-12 and inhibition of the UVB-induced expression of the Th2 cytokine IL-10. In this study, we seek evidence for an immunological waveband interaction when UVA and UVB are administered concurrently to hairless mice as occurs during sunlight exposure in humans. A series of spectra providing varying ratios of UVA/UVB were developed, with the UVA ratio increased to approximately 3.5 times the UVA component in solar simulated UV (SSUV). We report that progressively increasing the UVA component of the radiation while maintaining a constant UVB dose resulted in a reduction of both the erythema/edema reaction and the degree of systemic immunosuppression, as measured as contact hypersensitivity. The UVA-enhanced immunoprotection was abrogated in mice treated with a specific HO enzyme inhibitor. UVA-enhanced radiation also upregulated the expression of cutaneous IFN-gamma and IL-12 and inhibited expression of both IL-6 and IL-10, compared with the activity of SSUV. The results were consistent with the previously characterized mechanisms of photoprotection by the UVA waveband alone and suggest that the UVA component of solar UV may have beneficial properties for humans.

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. Both growth hormone (GH) and the inflammatory cytokine interleukin 6 (IL-6) have been implicated in prostate cancer progression. Studies in other systems have shown that an increase in GH results in an increase in IL-6 also. The current study demonstrated a parallel spatial and temporal expression of GH and IL-6 in cells in prostate cancer glandular acina cells. This study cannot determine if this expression is coincidental or causative, but it seems likely that the increase in GH could induce the expression of IL-6, since this is the case in other tissues. Optimal labelling for IL-6 in our study was achieved with low pH, high temperature antigen retrieval.

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. METHODS AND RESULTS:Initial biopsy sections from 25 cases of SCC and 20 of KA were labelled with markers for both the initiation (the cytolytic receptor P2X7) and end-stage (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) of apoptosis, telomerase-associated protein (TP1) and the cell adhesion protein E-cadherin. As this was a retrospective study, the clinical outcome of each case was known. This resulted in a unique labelling pattern of each marker for SCC and KA, allowing a differential diagnosis between the two conditions. The simplest marker to use for this purpose was anti-P2X7. Sections from five cases that were initially very difficult to diagnose were correctly identified as SCC using this method. CONCLUSIONS:These results support the view that KA has a different pathogenesis and biochemistry from that of SCC, and is a distinct entity. Anti-P2X7 labelling, using routine immunohistochemical techniques, provides a method for differentially diagnosing these conditions.

The cadherin family of proteins is significantly deexpressed in endometriosis and even more so in endometrioid adenocarcinoma. A similar finding was observed for the cadherin-associated cell adhesion protein plakoglobin. E-cadherin was not de-expressed in endometriosis but was significantly reduced in cancer. P-cadherin, a marker for dysplasia and neoplasia, was significantly increased in both endometriosis and cancer. Taken together, these findings suggest that cell adhesion is lost as dysplasia/neoplasia develops. It further suggests that endometriosis has features in common with endometrioid adenocarcinoma and perhaps with other cancers. (The J Histotechnol 28:185, 2005)Submitted: February 9, 2005; accepted July 11, 2005

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.

In the current study, expression of the apoptotic calcium channel receptor P2X 7 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 P2X 1 and P2X 2 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, P2X 1 and P2X 2 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 P2X 7 yielded similar results to that of P2X 1 and P2X 2 . 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 P2X 7 labeling results. All patients who exhibited no P2X 7 labeling had a prostatic serum antigen (PSA) level of <2. Patients who exhibited stage-specific P2X 7 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.

Aims: To detect early prostate cancer reliably by monitoring the expression of non-functional P2X(7) cytolytic purinergic receptors.Methods and results: P2X(7) receptors were absent from normal prostate epithelium obtained from post mortem tissue and tissue from cases of transurethral resection collected from young men (n = 23) who were confirmed to be free of cancer at later procedures 5-10 years after collection of the original samples. However, P2X(7) was present in every case of 116 confirmed prostate cancers regardless of Gleason grade or patient age. P2X(7) was present in apparently normal epithelial cells in acini well outside the tumour margins, but appeared in a distinct stage-specific manner commencing with the nucleus, progressing to the cytoplasm and collecting finally on the apical membrane of the epithelial cells in morphologically distinct cancer. The pattern of P2X(7) receptor localization in the epithelial cells was recorded in earlier biopsies obtained from the same patient cohort. One hundred and fourteen of 116 prostates stained positively for P2X(7) at the earliest biopsy, though generally with a less advanced pattern of distribution.Conclusions: The appearance of P2X(7) receptors in normal prostate tissue adjacent to prostate tumours makes direct tumour biopsy less critical for positive cancer diagnosis and enables cancer progression to be monitored.

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.

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 © 2003 John Wiley & Sons, Ltd.

Extensive labelling for the apoptotic markers calcium channel receptor P2X(7) and caspase-3 and telomerase activity was co-localized at a similar intensity in areas affected by superficial spreading melanoma obtained from 80 patients. Labelling for each of these markers also extended 2 microm from the melanoma into the keratinocyte layer of the adjacent normal epidermis. Conversely, the calcium-regulating receptors P2X(1-3) and P2Y(2) (found in normal but not neoplastic skin) were fully de-expressed within 2 microm of the melanoma but fully expressed beyond that distance. The cell adhesion protein E-cadherin (also only present in normal skin) was progressively de-expressed from a point 2 microm from the melanoma until full de-expression within the lesion. These results show that telomerase-induced proliferation and defensive apoptosis are co-localized and simultaneous processes in melanoma tissue. Melanoma cell proliferation appears to overwhelm the apoptotic defence, perhaps due to the anti-apoptotic effects of telomerase. In addition, keratinocyte regulation of the epidermis and dermis is severely compromised by the loss of E-cadherin and P2X(1-3) and P2Y(2) receptors, resulting in a lesion that is aggressive and malignant.

We examined the expression of the metabotropic P2Y(1), P2Y(2), P2Y(4), and ionotropic P2X(7) purinergic receptor subtypes in the uterine epithelium during early pregnancy in the rat. On Day 1 of pregnancy, there was no expression of P2X(7), P2Y(2), or P2Y(4) in the uterine epithelium. P2Y(1) was detected only as a diffuse label. On Day 3, P2X(7) and P2Y(2) receptor distribution was confined to the lateral plasma membranes in the epithelium. There was no expression of P2Y(4) while P2Y(1) was again detected only as a diffuse label throughout the epithelium. At the time of implantation on Day 6, a strong, continuous and area-specific P2X(7) and P2Y(2) label was noted along the entire surface of the apical epithelium suggesting a major role in calcium-modified events preceding and facilitating attachment and implantation of the blastocyst. P2Y(1) and P2Y(4) were present as a ubiquitous and nonspecific label, although the latter exhibited a minor apical deposition. These and earlier experiments with P2X subtype-specific antibodies indicate that both P2X and P2Y purinergic receptors play a role in conditioning the entire uterine epithelium for blastocyst implantation regardless of the site of attachment.

In mice fed a diet supplemented with red clover isoflavones the prostatic epithelium displays a significant increase in the production of estrogen receptor β and the adhesion protein E-cadherin but a decrease in transforming growth factor β1. These proteins are estrogenically-induced markers of proliferation, maintenance of histological architecture, preservation of cell phenotype and reduction of the potential for neoplastic and metastatic transformation. This study suggests that red clover isoflavones represent a non-toxic dietary treatment for prostatic hyperplasia and a reduction in the potential for neoplastic transformation.

The calcium-activated cell-adhesion proteins tenascin, E-cadherin and the purinergic (P2X) calcium channel receptors are expressed in an identical spatial and temporal pattern in uterine epithelium in the rat during implantation. On Day 1 of pregnancy (estrous), a diffuse cytoplasmic and specific basement membrane label for each of the proteins was observed throughout the uterine epithelium. On Day 3 of pregnancy, a specific and prominent lateral plasma membrane label for each protein was seen. At the time of implantation on Day 6, an additional and significant increase in the label for each was observed on the apical epithelium. At this time, the label for tenascin in the apical epithelium was increased 2.1-fold (p < 0.0004), that of E-cadherin was increased 2.5-fold (p < 0.0001) and the P2X receptor label was increased 2.0-fold (p < 0.0001). These observations suggest a major role for the calcium-activated adhesion proteins tenascin and E-cadherin in attachment and implantation, with ionic calcium for protein activation possibly provided by the P2X calcium channels. These events occur along the entire length of the uterine epithelium in preparation for blastocyst adhesion.