Differential expression of retinoic acid receptor beta (RARbeta) and the AP-1 transcription factor in normal, premalignant and malignant human laryngeal tissues.

The anticancer effects of retinoids are mainly mediated by their nuclear receptors. Recent studies have demonstrated that retinoic acid receptor beta (RARbeta) plays a pivotal role from the early stages of laryngeal carcinogenesis; however, the exact mechanism of this detrimental effect has not yet been elucidated. One of the best-documented actions of retinoid receptors is the transrepression of activator protein-1 (AP-1) transcription factor activity, although this complex interplay has not been clarified. The present report is the first systematic morphological evaluation of the cross-talk of RARbeta and AP-1 transcription factor in a large series of human laryngeal tissues containing normal epithelium, premalignant lesions (hyperplasia and/or dysplasia) and squamous cell carcinoma. Immunohistochemical methodology was performed on formalin-fixed, paraffin-embedded sections by using a panel of monoclonal and polyclonal antibodies against RARbeta and the AP-1 components c-Jun, p-c-Jun (phosphorylated, active c-Jun) and c-Fos proteins. Their expression was screened and compared in 154 patients with various laryngeal histological entities. Nuclear expression of RARbeta, c-Jun, p-c-Jun and c-Fos was detected in 81 (89.2%), 48 (52.8%), 66 (72.6%) and 73 (80.3%), respectively, out of 91 specimens with normal-appearing laryngeal epithelium; in 86 (87.8%), 94 (95.9%), 94 (95.9%) and 94 (95.9%), respectively, out of 98 specimens with hyperplastic laryngeal epithelium; in 58 (56.8%), 92 (90.2%), 96 (94.1%) and 96 (94.1%), respectively, out of 102 specimens with dysplastic laryngeal epithelium; in 10 (22.3%), 41 (91.2%), 44 (97.8%) and 41 (91.2%), respectively, out of 45 specimens with well-differentiated squamous cell carcinoma; in 13 (30.3%), 37 (86%), 39 (90.7%) and 41 (95.3%), respectively, out of 43 specimens with moderately-differentiated squamous cell carcinoma; and in 8 (66.7%), 10 (83.3%), 12 (100%) and 12 (100%), respectively, out of 12 specimens with poorly-differentiated squamous cell laryngeal carcinoma. Statistical analysis and correlation of the intensity of nuclear immunostaining of the studied proteins among the various histological entities revealed statistically significant results. The progressive upregulation of the AP-1 transcription factor constituents and downregulation of the RARbeta protein detected from the onset of laryngeal tumorigenesis suggests an important role for the immediate-early AP-1/RARbeta on/off "switch" in the process of laryngeal carcinogenesis.

Androgen receptor (AR) expression is an independent unfavorable prognostic factor in gastric cancer.

PURPOSE: To investigate the expression of sex steroid receptors in gastric cancer and to correlate their tumor expression profile with the clinicopathological parameters and overall survival of the patients. METHODS: Immunohistochemical methodology was employed in formalin-fixed paraffin-embedded sections from 86 patients with gastric carcinoma. Monoclonal antibodies against androgen (AR), estrogen (ER), and progesterone (PR) receptors were used. Survival rates were estimated by the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed by the Cox proportional hazards model. RESULTS: Fifteen (17.4%) cases of gastric adenocarcinomas were positive for AR, two (2.3%) were positive for PR and three (3.5%) were positive for ER. Significantly higher AR expression was found in tumors with metastases to lymph nodes (P = 0.03). Patients with AR-positive tumors (AR+) had worse prognosis than (AR-) patients (median survival 9 months vs 24 months, P = 0.03). Patients with AR- and heat shock protein 27 (HSP27)-positive tumors (AR+/HSP27+) had a median survival of 6 months, whereas (AR-/HSP27-) patients had a median survival of 42 months (P = 0.017). Multivariate analysis revealed that AR expression and UICC stage were independent factors of unfavorable prognosis (P = 0.037 and P = 0.0055, respectively). CONCLUSIONS: Identification of AR-positive gastric carcinomas in gastric biopsies may warrant a more aggressive therapeutic approach and anti-androgen or AR-targeted agents may represent a novel strategy in tackling this devastating malignancy.

Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation.

Oestrogen Receptor beta (ERbeta) may protect against prostate and mammary cell proliferation and malignant transformation. Epidemiological studies indicate that oestrogens may reduce colon cancer risk. Since ERalpha is minimally expressed in normal and malignant colon, the aim of this study was to investigate the expression of ERbeta in both normal colonic wall and colon cancer. ERbeta expression was evaluated by immunohistochemistry in 90 cases of colon adenocarcinoma and nearby (>30-cm away) normal colonic wall, using a monoclonal antibody. Moderate or strong nuclear immunostaining was detected in superficial and crypt epithelium, endothelial cells, vascular smooth muscle cells, lymphocytes, enteric neurons and smooth muscular cells of the normal colonic wall. Superficial epithelial cells in normal colon demonstrated a significantly higher ERbeta expression than colon adenocarcinoma cells in both genders. The decline in ERbeta expression paralleled the loss of differentiation of malignant colon cells, regardless of the tumour's localisation. These findings suggest a protective role for ERbeta against colon carcinogenesis.

Activation of the JNK-AP-1 signal transduction pathway is associated with pathogenesis and progression of human osteosarcomas.

Osteosarcomas represent the most common primary malignant bone tumors; however, comprehension of the molecular mechanisms underlying their pathogenesis is far from thorough. Studies in cultured cells have demonstrated that the c-Jun N-terminal kinase (JNK) signal transduction pathway participates in the proliferation, differentiation, and apoptosis of osteoblasts. Phosphorylated JNKs activate the oncoprotein c-Jun, which is known to form the activator protein-1 (AP-1) transcription factor as a homo- or heterodimer. c-Jun's principal dimerization partner is c-Fos, which participates in the differentiation and function of osteoblasts and in the pathogenesis of osteosarcomas. A similar role for the JNK cascade in the malignant transformation of human osteoblasts and in the generation of osteosarcomas has not been documented. Our study addressed the possibility that a functional upregulation of the JNK pathway is implicated in the pathogenesis of osteosarcomas. To this end, we employed immunohistochemistry to examine normal bone and osteosarcoma cells in paraffin-embedded sections from 56 patients with high-grade tumors and 15 patients with low-grade tumors. We assessed the protein levels of the two major JNK isoforms (JNK1 and JNK2); their phosphorylated-hence activated-species, p-JNK; their substrate, c- Jun; its phosphorylated (activated) form, pc-Jun; and c-Jun's heterodimeric partner, c-Fos. We also examined the immunohistochemical profile of the alpha chain of the nascent polypeptide-associated complex (alpha-NAC), an osteoblast-specific AP-1 coactivator that potentiates the transcriptional activity of the c-Jun/c-Jun homodimer. Positive immunostaining for JNK1, JNK2, p-JNK, c-Jun, pc-Jun, c-Fos, and alpha-NAC was observed in 86, 93, 94, 99, 97, 99, and 97.5% of the samples, respectively, whereas normal bone was devoid of these immunoreactivities. The cellular levels of all proteins were significantly correlated to each other (P < 0.001 for each correlation). Moreover, significantly higher expression levels of all proteins were detected in high-grade tumors compared to levels in low-grade ones. The observed expression profile of alpha-NAC implies that the active AP-1 in human osteosarcomas most likely comprises c-Jun/c-Jun homodimers. When cellular levels of the JNK pathway components and c-Fos were evaluated as possible biological markers of tumor grade, high expression of c-Jun and abundant pc-Jun predicted a high-grade tumor. Our findings provide novel evidence that the JNK signaling pathway is functionally operative in the malignant transformation of osteoblasts and the subsequent development and progression of human osteosarcomas. Evaluation of c-Jun expression and JNK-dependent activation may facilitate an improved prediction of the tumor's clinical behavior and potentially be exploited in designing patient-tailored treatment regimens.

Induction of the CBP transcriptional co-activator early during laryngeal carcinogenesis.

PURPOSE: CREB-binding protein (CBP) is a transcriptional "integrator" that is suspected of contributing to tumorigenesis. This is the first systematic morphologic study evaluating CBP expression in a large series of human laryngeal tissues containing normal epithelium, premalignant lesions (hyperplasia and/or dysplasia), and squamous cell carcinoma. METHODS: Immunohistochemical methodology was performed on formalin-fixed paraffin-embedded sections by using a monoclonal anti-CBP antibody. CBP expression was screened and compared in 156 patients with various laryngeal histologic entities. RESULTS: Nuclear expression of CBP was found in 44 out of 91 (48.4%) specimens with normal-appearing epithelium (46.2% weak and only 2.2% moderate positivity), 92 out of 100 (92%) with hyperplastic lesions (56% weak, 36% moderate/strong, and only 8% no positivity), 80 out of 103 (77.7%) with dysplastic lesions (45.6% weak, 32.1% moderate/strong, and 22.3% no positivity), 37 out of 45 (82.2%) with well-differentiated carcinoma (42.2% weak, 40% moderate/strong, and 17.8% no positivity), 31 out of 43 (72.1%) with moderately differentiated carcinoma (32.6% weak, 39.5% moderate/strong, and 27.9% no positivity) and eight out of 12 (66.7%) with poorly differentiated carcinoma (41.7% weak, 25% moderate/strong, and 33.3% no positivity). Statistical analysis and correlation of the intensity of nuclear immunostaining among the various histologic entities revealed statistically significant results. CONCLUSIONS: Overexpression of CBP is detected from the very early stages of laryngeal carcinogenesis, suggesting that CBP may play a role in malignant transformation of precancerous laryngeal lesions. It is possible that overexpression of this protein is a prerequisite for the observed p53 upregulation in premalignant lesions, implying an indirect role of CBP in p53-mediated tumorigenic potential.

Cellular distribution of retinoic acid receptor-alpha in benign hyperplastic and malignant human prostates: comparison with androgen, estrogen and progesterone receptor status.

OBJECTIVES: Retinoids are unique modulators of gene activity, cell growth and differentiation by binding to a series of nuclear receptors, i.e. all-trans-retinoic acid receptors (RAR) or 9-cis-retinoid receptors (RXR). In this study, the expression of RARalpha was immunohistochemically evaluated in benign, hyperplastic and malignant prostatic tissue and correlated with sex steroid receptor status. METHODS: Twenty-four cases of BPH and 139 cases of primary prostatic carcinoma were evaluated for RARalpha expression in correlation with androgen (AR), estrogen (ER) and progesterone (PGR) receptor staining, as well as with tumor grade. RESULTS: RARalpha was detected in the nuclei of epithelial cells in both BPH and prostate carcinoma cases. A modest inverse relationship with grade was present, especially for grade I and grade II tumors. AR staining was intense and a strong inverse relationship with grade was revealed. Although ER and PGR showed nuclear staining in prostatic epithelium, the overall expression for these receptors was low. When RARalpha content was compared to the nuclear AR expression, at least two-fold higher RARalpha levels were observed in AR+ grade II and grade III tumors. CONCLUSIONS: RARalpha expression can be immunohistochemically evaluated in formalin-fixed paraffin-embedded prostatic tissue. RARalpha expression is significantly elevated in AR+ moderately and poorly differentiated prostate carcinomas. Immunohistochemical determination of RARalpha content may be useful in defining the patient subsets in which retinoid-based treatment may be of clinical value.

Immunohistochemical detection of retinoic acid receptor-alpha in prostate carcinoma: correlation with proliferative activity and tumor grade.

BACKGROUND: The use of retinoids as differentiation therapy is a novel approach to prostate cancer. Retinoids act via their own nuclear receptors, RARs and RXRs, modulating gene activity, cell growth and differentiation. This study provides new data on the content and cellular distribution of RARalpha protein in prostate cancer specimens, in correlation to tumor grade and proliferative activity. MATERIAL AND METHODS: A total of 84 cases of primary prostate carcinoma, divided into 3 subgroups according to tumor grade, were immunohistochemically evaluated for retinoic acid receptor-alpha (RARalpha) and Ki67 using the streptavidin/peroxidase method on formalin fixed, paraffin embedded tissues. RESULTS: RARalpha positivity was detected in all cases of prostatic carcinoma, with a more profound expression in well differentiated cancers. A statistically significant correlation between RARalpha staining and tumor grade was found (ANOVA, p < 0.031). Ki67 immunoreactivity was present in 35.7% of cases, but no correlation with tumor grade was found. When RARalpha staining was correlated with Ki67 positivity, a statistically significant correlation was present (unpaired t-test, p < 0.003). CONCLUSIONS: These findings indicate that RARalpha expression is correlated to some extent with tumor grade and its presence is more profound in highly proliferative tumors. Further studies are needed to establish the possible clinical value of the immunohistochemical evaluation of RARalpha content in tumor specimens.

Prognostic significance of Hsp-27 in astrocytic brain tumors: an immunohistochemical study.

Formalin-fixed paraffin-embedded tumor specimens from 95 patients with supratentorial astrocytic brain tumors were immunostained by a monoclonal antibody against the heat shock protein-27 (Hsp-27) using the streptavidin/peroxidase method. The immunohistochemical analysis was scored in a semiquantitative fashion incorporating both the intensity and distribution of specific staining (score): the immunohistochemical results were correlated with the histological grade of the tumors and patients' sex and age. Furthermore, Hsp-27 expression was studied in two groups into which the patients were further divided: group (a) previously untreated patients (n = 76) whose biopsy or surgical resection was related to their initial presentation and diagnosis and group (b) patients (n = 19) with reccurent disease who underwent radiotherapy and/or chemotherapy. Strong Hsp-27 cytoplasmic immunopositivity was observed in 42 out of 51 (82%) glioblastomas, in 8 out of 20 (40%) anaplastic astrocytomas and in 2 out of 24 (8%) astrocytomas. The mean Hsp-27 score was 45.2 in glioblastomas, 6.5 in anaplastic astrocytomas and 0.4 in astrocytomas. The expression of Hsp-27 immunoreactivity appeared to be independent of the age and sex of the patients. A non significant difference was defined between untreated patients and previously treated patients. Hsp-27 immunoreactivity was observed in the microvascular endothelial proliferations and in tumor blood vessels. Normal astrocytes were Hsp-27 negative. These findings indicate that, in contrast with the low Hsp-27 expression found in benign astrocytomas, the expression of Hsp-27 in a number of poorly differentiated tumors, including glioblastomas and anaplastic astrocytomas, is consistent and independent of previous treatment of the patients. We support the involvement of Hsp-27 in the growth of astrocytic brain tumors.

Microvessel density in brain tumors.

Sections from formalin fixed paraffin embedded tumor tissue from 165 patients with brain tumors including 62 meningiomas, 80 supratentorial astrocytomas (19 astrocytomas-grade I/II, 20 anaplastic astrocytomas-grade III, 41 glioblastomas-grade IV), 7 cerebellar astrocytomas-grade I/II, one gliosarcoma, 7 oligodendrogliomas, 3 ependymomas and 5 medulloblastomas were immunostained for factor VIII-related antigen in order to highlight microvessel endothelial cells. Microvessel count (MVC; the highest number of microvessels in three areas of highest vascular density at X200 magnification) was determined and correlated with histological grade of tumors and patients' sex and age. The mean MVC was 27.9 in meningiomas. Astrocytic tumors, particularly malignant astrocytomas (grade III, IV), were highly vascular. The mean MVC as regards the supratentorial astrocytic neoplasms was 14.5 in astrocytomas (grade I/II), 42.3 in anaplastic astrocytomas (grade III) and 50.2 in glioblastomas (grade IV). All cerebellar astrocytomas studied, even though well differentiated (grade I/II), were highly vascularised tumors (MVC: 41.1. A comparison of the mean MVC vlaues showed that there was a statistically significant difference between supratentorial astrocytomas (Grade I-II) and cerebellar astrocytomas (Grade I-II) (p = 0.0004), anaplastic astrocytomas (Grade I-II) (p = 0.00004) and glioblastomas (p = 0.00001). There was no significant difference between cerebellar astrocytomas and anaplastic astrocytomas (p = 0.8) and glioblastomas (p = 0.4). Astrocytic neoplasms showed statistically significant higher mean MVC from meningiomas (p = 0.002). The mean MVC was 14.1 in oligodendrogliomas, 22.7 in ependymomas and 19.6 in medulloblastomas. In one gliosarcoma that was studied the MVC was 40. The MVC appeared to be independent of the age and sex of patients. This study supports the importance of microvessel density as a measure of angiogenesis, as well as a further morphologic feature in the classification of brain tumors. The determination of microvessel density may become useful in the planning and monitoring of anti-angiogenesis therapies of these tumors.

[Histologic study of corneal lesions caused by the slime-GLP glycolipoprotein of Pseudomonas aeruginosa]. / Etude histologique des lésions cornéennes créées par la glycolipoprotéine Slime-GLP du Pseudomonas aeruginosa.

The Pseudomonas aeruginosa slime-glycolipoprotein (GLP) is considered as one of the principal pathogenetical factors of the bacterium. A single dose of 100 micrograms of the P. aeruginosa slime-GLP was injected in rabbit corneas intrastromally. Light microscopy showed that 4 hours after the injection, polymorphonuclear leucocytes (PMNs) began to infiltrate the anterior stroma. 24 hours after the intrastromal injection, PMNs had infiltrated full corneal thickness followed by multiple absceses formation, loss of epithelial and endothelial cells, disorganisation of normal collagen fibres and hyperplasy of fibroblasts. These morphological observations are very similar to those observed during experimental P. aeruginosa keratitis and show that the P. aeruginosa slime-GLP is at least in part responsible for the characteristic liquefaction necrosis of the keratitis induced by the P. aeruginosa.