Distant metastases from prostatic carcinoma express androgen receptor protein.

Nearly all primary prostatic carcinomas have been found to express the androgen receptor (AR) protein, which is the intracellular mediator of androgen action. To gain a better insight into the mechanisms of androgen independence of advanced prostatic carcinoma, it is important to know whether the AR is also present in metastases of androgen-independent tumors. We have assessed the status of the AR and the prostate-specific antigen in 22 metastases of 18 patients with progressive prostate cancer. In 18 cases, the metastases were localized in bone, in 3 cases in the epidural space, and in 1 case in the periosteum. All but one patient had received some kind of endocrine treatment for prostatic carcinoma. Paraffin-embedded tissue sections were stained for the AR following a streptavidinbiotin-peroxidase protocol with the polyclonal antibody PG-21, which is directed against amino acids 1 through 21 of the rat and the human AR. The percentage of AR-positive cells was evaluated on the basis of an arbitrary 4-point scale. All 22 tumor metastases displayed AR positivity. One AR-positive metastatic lesion did not stain for prostate-specific antigen, but in all other metastases, this protein was detected by means of immunohistochemistry. The present study provides evidence that, unlike androgen-independent prostatic carcinoma cell lines, distant prostatic carcinoma metastases do express the AR. These findings indicate that the AR may be involved in the progression of prostate cancer.

Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor.

Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.

Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone.

Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.

HIV-1 and HIV-2 isolates differ in their ability to activate the complement system on the surface of infected cells.

OBJECTIVE: To analyse the ability of different HIV-1 and HIV-2 isolates to activate the complement system. DESIGN: H9 cells chronically infected with various HIV isolates and the corresponding purified viruses were tested for complement activation. To identify the molecules responsible for complement activation on the surface of infected cells, the expression of complement inhibitors/regulators and viral proteins on the cell surface was analysed. METHODS: C3 deposition on the cell surface and the expression of viral and cellular antigens were determined by flow cytometry analysis. Complement activation by purified viruses was measured using a complement consumption assay and a C1 activation assay. RESULTS: H9 cells infected with different HIV-1 and HIV-2 isolates showed varying degrees of complement activation on the cell surface, ranging from strong activation and deposition of large amounts of C3 to no increased C3 deposition compared to uninfected cells. The C3 deposition was eliminated by EDTA and reduced in the presence of EGTA. In contrast, all purified viral isolates tested activated the complement system in a comparable manner. While the expression of MCP, DAF and CD59 was not modified after infection with different viral isolates, the reaction of the infected cells with a monoclonal antibody (3D6) directed against a gp41 epitope (amino acids 601-620) was found to correlate with the complement activation on the cell surface. CONCLUSIONS: Some HIV-1 as well as HIV-2 isolates activate the complement system on the surface of infected cells independent of anti-HIV antibodies, while other isolates fail to do so. Complement activation on the cell surface is mediated by the alternative and, to a lesser extent, the classical pathway. The differences in complement activation on the cell surface are not caused by a modified expression of membrane-bound complement inhibitors/regulators. C3 deposition on the cell surface correlates with the expression of an epitope lying within the major complement activating domain of gp41 (amino acids 591-620). These results suggest a role of gp41 for complement activation on HIV-infected cells as has been described previously for purified HIV.

Expression of the human chorionic gonadotropin-beta gene cluster in human pituitaries and alternate use of exon 1.

Previous studies have indicated that in addition to other glycoprotein hormones, the pituitary gland produces small amounts of hCG beta, the classical pregnancy and tumor marker. At the gene transcription level, definitive proof for hCG beta messenger ribonucleic acid transcription was still lacking, largely due to the 90% homology to hLH beta at the DNA sequence level, which renders specific hCG detection in the presence of a vast excess of LH difficult. We investigated both the presence of hCG beta messenger ribonucleic acid and the protein itself in normal human female postmenopausal (n = 4) and male pituitaries (n = 2). Reverse transcription-PCR and subsequent restriction enzyme analysis revealed that the hCG beta 3, 5, 7, and 8 genes coding for genuine hCG beta were transcribed in all pituitaries. Additionally, three alternatively spliced gene products derived from hCG beta genes 1 and 2 were detected and verified by single strand sequencing of the complementary DNAs. The most abundant fragment (244 bp) showed a point mutation (T-->A) in the splice donor site for the first intron, resulting in an alternate use of exon 1 and a frame shift in the open reading frame that might give rise to a hypothetical protein, 132 amino acids in length. With regard to protein synthesis, we confirmed the pituitary as the site of production for hCG beta by reverse phase high performance liquid chromatography and subsequent immunoradiometric assays, including a monoclonal antibody directed against the unique C-terminal extension of hCG beta.

Inhibition of endogenous peroxidase for the immunocytochemical demonstration of intermediate filament proteins (IFP).

The necessity for minimally fixed and processed cell and tissue preparations for immunocytochemical studies of sensitive antigens such as lymphocyte surface markers is well recognised. In order to avoid methanol and hydrogen peroxide, which have been shown to be deleterious for certain antigens, various compounds have been proposed for blocking endogenous peroxidase activity (EPA) in tissue preparations which are to be used in immunoperoxidase reactions. In the present study the deleterious effect of methanol/H2O2 on intermediate filament proteins was demonstrated in both frozen sections and paraffin-embedded tissue. The use of alternative reagents for the non-deleterious blocking of EPA is recommended for immunocytochemical staining with antibodies against intermediate filaments.

Chromogranin A and B in adenomas of the pituitary. An immunohistochemical study of 42 cases.

Forty-two pituitary adenomas (10 prolactinomas; three ACTH-, nine GH-, two FSH- and two TSH-secreting adenomas; and 16 clinically nonfunctioning null cell adenomas) were investigated immunohistochemically with antibodies against chromogranin A and B as well as ACTH, GH, prolactin (PRL), FSH, LH, TSH, and alpha-HCG antibodies. For the demonstration of chromogranin B, two different antibodies were used--e.g., a polyclonal antihuman antibody and an antiserum against a synthetic peptide (DK-21, chromogranin B 306-326) present in the chromogranin B amino acid sequence. All tumors were positive for both chromogranin B antibodies. Chromogranin A was found in FSH- (two of two) and TSH- (two of two) secreting adenomas; it was also found in a focal distribution in ACTH- (one of three) and GH- (four of nine) secreting adenomas. Thirteen of 16 null cell adenomas contained chromogranin A, whereas no chromogranin A was found in prolactinomas. We conclude that null cell adenomas may arise either from FSH/LH or TSH cells (null cell adenomas with both chromogranin A and B positivity) or from ACTH, GH, or PRL cells (the respective tumors are only positive for chromogranin B). Chromogranin B may be used as a universal marker for pituitary adenomas.

Gemcitabine--a novel immunosuppressive agent--prevents rejection in a rat cardiac transplantation model.

BACKGROUND: 2',2' -difluorodeoxycytidine (dFdC, gemcitabine) is a pyrimidine antimetabolite with antineoplastic activity against a wide range of solid tumors. The immunosuppressive activities of this compound have not been described to date. METHODS: The in vitro effects on activated T lymphocytes were studied with a lymphocyte colony-forming assay in a microagar culture system. Heart transplantations were performed in the fully allogeneic Lewis/ Brown Norway combination. dFdC was administered once daily at various dosages from the time of surgery until day 50. RESULTS: Phytohemagglutinin-induced lymphocyte proliferation was inhibited 50% by dFdC at a concentration of 3.25+/-0.9 nmol/L. Allografts of untreated animals survived for 7.5 (7-8) days and those with 25, 50, and 75 microg/kg body weight dFdC for 7.3 (7-8), 9.3 (8-10), and 16.3 (10-38) days, respectively. Treatment with 100 or 125 microg/kg body weight of dFdC, however, prolonged allograft survival until day 152.8 (129-178). Dose-dependent leukopenia was the main toxicity. CONCLUSIONS: DFdC is a new immunosuppressive agent that can successfully prevent cardiac rejection in a rat transplantation model.

Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach.

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.

Quantitative immunohistochemical evaluation by image analysis of metallothionein in copper-loaded rat kidney.

Kidneys of copper-loaded rats were investigated immunohistochemically with a directly peroxidase-conjugated monoclonal antibody against metallothionein (MT). By means of an image analyzing system the area and the staining intensity of MT immunoreactivity in the proximal convoluted tubule (PCT) cells were determined. In the present study we compared the data obtained by image analysis with analytically determined tissue copper and MT concentrations of rats fed a high-copper diet (1 g/kg) for 16 weeks and sacrificed sequentially during this period. Our results provide evidence that the area of MT immunoreactivity correlates significantly with tissue copper and MT concentrations. Both the copper and MT concentrations in kidney rose to a maximum at 8 weeks and remained constant thereafter. The observed rise in the staining intensity of MT in PCT cells to a maximum at 6 weeks, which subsequently declined, suggests a continuing redistribution of copper and MT in the kidneys even after a maximum of concentration copper and MT is reached in the tissue.

Characterisation of a collecting duct carcinoma by cytogenetic analysis and comparative genomic hybridisation.

Collecting duct carcinoma (CDC) is a rare variant of carcinoma of the kidney with aggressive behaviour. CDC arise from the epithelium of the ducts of Bellini in the distal nephron. A CDC in a 53-year old male patient is characterised both by cytogenetic analysis and comparative genomic hybridisation (CGH). The cytogenetic analysis shows a biclonal karyotype 47,XY,+3[2]/40-44,XY,-12[3], -22[4][cp5]/46,XY[42]. Loss of DNA sequences detected by CGH involves chromosome 1, 2, 9, 11 and 18. Gain of DNA sequences affect chromosome 16 and 20. The characterisation of a CDC in this study represents an additional contribution to the poorly explored CDC.

Combined study of prostatic carcinoma by classical cytogenetic analysis and comparative genomic hybridization.

Conventional cytogenetic analysis of prostatic carcinoma (PC) is characterized by inefficient growth of tumor cells during in vitro culture, leading to a lack of aberrant karyotypes in many of investigated tumors. In this study we have combined a modified short-term tissue culture method for conventional banding analysis and comparative genomic hybridization (CGH) to examine genetic changes in PC, and to evaluate the effect of the in vitro culture on chromosomal changes by comparing results of the two methods. Cytogenetic analysis was performed on 34 PCs using both, conventional and molecular methods. Tumor tissues were obtained predominantly from untreated primary tumors from 48 patients. For karyotyping all tumor samples were short-term cultured using a feeder layer technique. Additionally DNA from uncultured tumor material from 17 of those patients was isolated and screened for copy number changes using CGH. Conventional banding analysis: clonal aberrations were detected in 65% of the tumor samples. Most of the chromosomal findings were numerical changes, including loss of chromosomes Y (32%), 18, 19 and 21 (each 12%). Less frequent, trisomy of chromosome 7 and monosomy of chromosomes 9, 12 and 22 (each 9%) was found. Additionally an inversion of chromosome 9p and a deletion at chromosome 7q was found in two cases. In 35% no clonal aberrations could be detected. CGH: DNA copy number changes were detected in 65% of the analyzed tumors. Predominantly losses of DNA sequences were found. The most common losses were found at chromosome regions 13q21q33 (29%), 6q11q23 (24%), 16q, and 18 (each 18%), and the most common gains at 19 (18%). In six tumors no copy number changes were found. Both methods showed a similar aneuploidy rate, suggesting that the feeder layer technique is quite a suitable method for in vitro culture of PC cells. However, the two techniques produced substantially differing results for most of the tumor samples, and in some cases the discrepancies are quite striking. Therefore eventual culture effects need to be taken into account when comparing results from conventional cytogenetics and CGH. Some contrary findings from the two methods are discussed.

CD30 expression in seminoma.

In testicular germ cell tumors the CD30 antigen has been shown to be regularly expressed in embryonal carcinoma and was thus suggested as a marker for this particular neoplasm. Very recently, it has been proven that the monoclonal antibody Ber-H2 is suitable for the detection of this membrane antigen in paraffin sections. We conducted an immunohistochemical study to investigate the CD30 expression in a large series of different presentations of seminoma (ie, pure, mixed, and spermatocytic) because there is evidence from several sources that embryonal carcinoma is histogenetically closely related to, and probably derives from, seminoma. Sections from formalin-fixed, paraffin-embedded tissue from 38 cases of testicular seminomas were immunostained for the demonstration of the CD30 antigen using the monoclonal antibody Ber-H2, cytokeratins, and placental alkaline phosphatase following an indirect streptavidin-peroxidase regimen. In selected cases, immunostainings were performed on consecutive sections to investigate a possible colocalization of CD30 and cytokeratins in seminoma. Specific immunostaining for CD30 in seminoma cells could be detected in single minute foci in 4 of 21 cases of pure classic seminoma. Seminomatous components of mixed tumors showed CD30 positivity in single, but also multiple, foci in 7 of 14 cases. CD30 immunoreactivity in seminoma cells occurred with and without colocalized expression of cytokeratin. Spermatocytic seminoma (n = 3) as well as intratubular germ cell neoplasia in tumor adjacent parenchyma (n = 36) were negative in all cases investigated. We conclude that in testicular germ cell tumors, the expression of CD30 is not restricted to embryonal carcinoma but can also be found focally in seminoma, adding further evidence for a close relationship between these two tumors. The prevalence of CD30 expression in seminomatous components of mixed tumors, as well as the coexpression with cytokeratins, suggest that CD30 expression in seminomas might indicate their upcoming transformation to embryonal carcinoma. This conclusion coincides with a model featuring seminoma in a central role of germ cell tumor development.

Ovarian Sertoli-Leydig cell tumor: a SRY gene-independent pathway of pseudomale gonadal differentiation.

Sertoli-Leydig cell tumors (SLCT) are rare sex-cord stromal tumors of the ovary composed of undifferentiated gonadal stromal cells, Leydig cells (LC), and Sertoli cells (SC), with the latter forming structures resembling fetal testicular tubules. The histogenetic basis of morphological male differentiation patterns in females is controversial. Here, we report a SLCT with intermediate differentiation in a 23-year-old woman investigated by light microscopy, immunohistochemistry for intermediate filaments, and sex steroid hormone receptors (SSHR), as well as by polymerase chain reaction (PCR) for the presence of the sex-determining region Y gene (SRY). Our investigation shows that the SCs of SLCT express progesterone and androgen receptors as well as cytokeratins and vimentin. By PCR, SLCT-derived genomic DNA lacked the SRY gene, indicating that the SLCT results from a SRY gene-independent pathway of pseudomale gonadal differentiation. The expression of progesterone receptors (PRs) in the SCs of the SLCT is in contrast to their absence in testicular SCs, but in line with their presence in ovarian granulosa and surface epithelial cells. Thus, our results provide strong evidence for a close histogenetic relationship between the SLCT and the female gonocyte-supporting cell, the granulosa cell (GC).

Immunohistochemical demonstration of chromogranins A and B in neuroendocrine tumors of the lung.

Fifty neuroendocrine tumors of the lung (16 carcinoids, two atypical carcinoids/well-differentiated neuroendocrine carcinomas [WDNCs], 13 neuroendocrine carcinomas of intermediate cell type [SCNCs], and 19 neuroendocrine carcinomas of small cell type [SCNs]) were immunohistochemically investigated with antibodies against chromogranins A and B. All carcinoids and WDNCs were positive for both chromogranins A and B, whereas in cases of ICNC and SCNC both markers were only expressed in six and five cases, respectively. One ICNC was only positive for chromogranin A. In cases of SCNC five tumors were exclusively positive for chromogranin A and six were positive only for chromogranin B. Chromogranins are therefore excellent markers for the immunohistochemical demonstration of carcinoids and WDNCs. It may be speculated that expression of chromogranins in cases of ICNC and SCNC represents a higher degree of differentiation in these tumors.

The testis-specific expression pattern of the growth hormone/placental lactogen (GH/PL) gene cluster changes with malignancy.

Growth hormone (GH) and placental lactogen (PL) gene transcription patterns in testicular germ cell tumors (GCT) and normal testicular tissue were comparatively investigated to identify GH/PL gene products associated with the development of GCT. This was done by nondiscriminative reverse transcriptase-polymerase chain reaction (RT-PCR), amplifying all major transcripts of any of the 5 GH/PL genes--GH-N(ormal), GH-V(ariant), PL-A, PL-B, PL-L(ike)--and subsequent analytical restriction enzyme analyses of 5'-end radioactively labeled cDNA. Surprisingly, all nonseminomatous GCT (NSGCT; n = 9) expressed GH-N, PL-A/B, and PL-L transcripts (9 of 9). Seminoma (n = 7) showed a distinctly unique pattern of GH-N and PL-A/B. GH-V products, which are hallmarks of the normal healthy testis, were not detected in any testicular cancer specimen (0 of 16). The fact that both seminomatous and NSGCT showed alterations in the same gene cluster indicates a pathogenetic relationship. Two choriocarcinoma cell lines of conceptus origin, BeWo and JAR, clearly differing from the male counterparts, exhibited a placental-derived pattern of PL-A/B and GH-V. Obviously, profound differences exist between conceptus and male germ cell GH/PL gene cluster transcription. In summary, the unique testicular pattern of GH/PL gene expression changes significantly and in directed ways with malignancy. Loss of GH-V gene expression in testicular GCT compared with normal testis and loss (seminoma) or mutation (NSGCT) of PLL gene products might have significance in terms of the relationship between these tumors and for testicular GCT development.

Analysis of Bcl-2 protein expression in chronic lymphocytic leukemia. A comparison of three semiquantitation techniques.

A number of studies revealed that high expression of the proto-oncogene bcl-2 correlated with poor prognosis or resistance to chemotherapy in some tumors but predicted a favorable clinical course in other neoplasias. In these studies, however, different immunologic techniques for Bcl-2 detection were used, raising the question of whether the methods applied were comparable. Using chronic lymphocytic leukemia (CLL) cells, the aims of our study were as follows: (1) to determine the reproducibility of Bcl-2 semiquantitation by immunocytochemistry, flow cytometry, or immunoblotting; (2) to study the agreement between results obtained by these methods; and (3) to examine the association between Bcl-2 expression in tumor cells of 99 patients with CLL and clinical parameters. We found that determination of Bcl-2 expression by immunocytochemistry was reproducible and the results were comparable with those of flow cytometry and immunoblotting. In the patient collective examined, Bcl-2 expression did not reflect the extent of tumor mass, but higher levels were found more often in patients with progressive disease.

Hyperactive androgen receptor in prostate cancer: what does it mean for new therapy concepts?

Investigations on androgen signaling alterations in the late stages of prostate cancer revealed new molecular mechanisms that may be in part responsible for failure of endocrine therapy. Both primary and metastatic lesions from prostate cancer express androgen receptor protein. Amplification of androgen receptor gene occurs in a subset of prostate cancer patients. Several point mutations of androgen receptor gene have been described; they generate receptors which are functionally activated by androgens, other steroids, and even by antihormones. The frequency of androgen receptor mutations may be high in tumor metastases. Functional activity of androgen receptor is influenced by nonsteroidal factors, such as peptide growth factors and second messengers. Thus, prostate cancer cells adapt to low androgen environment by various mechanisms utilizing androgen receptor. Therefore, new strategies for switching off the androgen receptor are needed.

Expression of vimentin, cytokeratin, and desmin in Sertoli cells of human fetal, cryptorchid, and tumour-adjacent testicular tissue.

The intermediate filament of mature human Sertoli cells is vimentin. A co-expression of vimentin together with cytokeratin has been demonstrated in Sertoli cells during embryonal development and under pathologic conditions in adult testes. We analysed the presence of vimentin, cytokeratin, and desmin in Sertoli cells of fetal testes (n=20), in seminiferous tubules of cryptorchid testes (n=10) and adjacent to testicular germ cell tumours (n=47) using specific monoclonal antibodies and single and double-labelling immunohistochemistry. During embryonal development prominent cytokeratin expression disappears after the 20th week of gestation. Interestingly, we also found desmin in immature intratubular Sertoli cells between weeks 11 and 14. In adult cryptorchid testes and in peritumour tubules, desmin was also prominently present in Sertoli cells in the vast majority of the cases investigated, as well as vimentin and cytokeratin co-expression. This first description of desmin immunoreactivity may shed some light on the ontogeny of human Sertoli cells and demonstrates that this cell type is able to express three types of intermediate filaments in a complex manner.

In vitro investigations of interphase and metaphase argyrophilic nucleolar organizer regions and cellular proliferation in the human urothelial cancer cell line HOK-1.

Detailed investigation of cell growth and nucleolar organizer region associated argyrophilic proteins (Ag-NORs) is necessary to assess a possible impact of Ag-NOR quantification on the diagnosis and prognosis of tumours. In this study, cellular proliferation of the transitional-cell carcinoma cell line HOK-1 was modulated over a period of 11 days by starvation and subsequent medium addition. Proliferation was determined daily by DNA flow cytometric estimation of S-phase fraction (SPF) and mitotic index (MI) calculation. The number and area of interphase Ag-NORs were quantified by automated image analysis daily and the number of Ag-NOR bearing chromosomes in metaphase was counted. In interphase nuclei, Ag-NOR area showed a highly significant correlation with SPF (p < 0.0001) whereas interphase Ag-NOR number showed significant correlation with MI (p < 0.05). A positive relationship between the number of Ag-NOR bearing chromosomes in metaphases and cellular proliferation was also observed. There is variability in Ag-NOR quantity during interphase and metaphase depending on growth conditions in vitro. Correlations of the number of interphase Ag-NORs with the MI on one hand and Ag-NOR area with SPF on the other provide further evidence that distribution and quantity of Ag-NORs are strongly influenced by the cell cycle phase within the structural-functional unit of the nucleolus.