Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18⁺/PSA^– HT29 colon carcinoma cells. CK18⁺ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.     

Immunological characterization of human acid phosphatase gene products

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10–20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not cross-react with the erythrocyte enzyme. (10–20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.This study was supported by DHHS Research Grant GM 27003 from the U.S. National Institute of General Medical Sciences and by Grant SFB-104 from the Deutsche Forschungsgemeinschaft.     

The Deceptive Acid Phosphatases

Determination of the prostatic acid phosphatase is, theoretically, a specific test for carcinoma of the prostate, but the present laboratory techniques have produced too many false positives and false negatives to be dependable. There may be inhibitors or enzymes that interfere with these tests.Until more exact enzymes are discovered, the present acid phosphatases should not be depended upon as a criterion for the type of surgical operation in carcinoma of the prostate, nor, without biopsy, should they be taken as an indication of prostatic malignant disease.     

Clinical significance of tartrate-sensitive and tartrate-resistant acid phosphatase indicated from the study of their biosynthetic mechanism

The tartrate-sensitive prostatic acid phosphatase, bands 2 and 4, are found in the soluble cytosol, and absent in the polysome of the prostate, while the tartrate-resistant acid phosphatase band 5 is present in the polysome and the soluble cytosol of hairy cells. The mRNA isolated from the prostate catalyzes the incorporation of 3T leucine into a protein different from that of bands 2 and 4. On the other hand, the mRNA isolated from the hairy cells catalyzes the incorporation of 3T leucine into band 5. The different biosynthetic mechanism of these two types of acid phosphatases are discussed in light of their different clinical significance.     

Intra-tibial injection of human prostate cancer cell line CWR22 elicits osteoblastic response in immunodeficient rats

We investigated the utility of CWR22 human prostate cancer cells for modeling human metastatic prostate cancer, specifically their ability to induce bone formation following intra-tibial injections in the nude rat. Prostate cancer is unique in regard to its tropism for bone and ability to induce new bone formation. In contrast to humans, other mammalian species rarely develop prostatic cancer spontaneously upon aging and do not have the propensity for bone metastasis that is the hallmark of cancer malignancy in men. We chose human prostate cancer cell line CWR22 based on its properties, which closely resemble all of the features that characterize the early stages of prostatic cancer in human patients including slow growth rate, hormone dependence/independence and secretion of prostate-specific antigen. When CWR22 cells were injected directly into the proximal tibia of immunodeficient male rats, both osteoblastic and osteolytic features became evident after 4 to 6 weeks, with elevated levels of serum prostate-specific antigen. However, osteosclerosis dominates the skeletal response to tumor burden. Radiological and histological evidence revealed osteosclerotic lesions with trabeculae of newly formed bone lined by active osteoblasts and surrounded by tumor cells. Toward the end of the 7-week study, osteolytic bone lesions become more evident on X-rays. Paraffin and immunohistochemical evaluations revealed mature bone matrix resorption as evidenced by the presence of many tartrate resistant acid phosphatase positive multinucleated osteoclasts. We conclude that the CWR22 human prostate cell line used in an intra-tibial nude rat model provides a useful system to study mechanisms involved in osteoblastic and osteolytic bony metastases. This type of in vivo model that closely mimics all major features of metastatic disease in humans may provide a critical tool for drug development efforts focused on developing integrated systemic therapy targeting the tumor in its specific primary or/and metastatic microenvironments. In addition to targeting bone marrow stroma, this strategy will help to overcome classical drug resistance seen at the sites of prostate cancer metastasis to bones.     

Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.     

Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.     

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.     

The isolation and identification of exfoliated prostate cells from human semen

A prostatic acid phosphatase localization technique to identify exfoliated prostate cells in human semen is described. Three morphologically distinct types of exfoliated prostate cells were observed in the semen of 14 vasectomized and nonvasectomized men: (1) large, ovoid prostate cells with abundant cytoplasm; (2) small, ovoid prostate cells with little cytoplasm; and (3) columnar prostate cells. These three types of exfoliated prostate cells were also observed within the lumina of prostatic alveoli when stained by this method. Finally, this staining procedure was found to have certain advantages over the conventionally used staining techniques, for it allows the rapid identification of exfoliated prostate cells in human semen and provides a noninvasive technique for monitoring the functional state of the prostate gland.     

Molecular Cloning of a Novel Human Acid Phosphatase Gene (ACPT) That Is Highly Expressed in the Testis

Acid phosphatases are enzymes capable of hydrolyzing orthophosphoric acid esters in an acid medium. Prostatic acid phosphatase has served as a tumor marker for metastatic prostate cancer for many years. We have cloned a new human acid phosphatase gene (named testicular acid phosphatase, ACPT), which is expressed mainly in testis and to a lower extent in the prostate, trachea, and other tissues. This gene maps to chromosome 19q13.4, in an area that harbors many cancer-related genes. The testicular acid phosphatase gene is composed of 11 exons, and the protein is predicted to have a luminal domain, a transmembrane domain, and a cytoplasmic domain. The N-terminal end of the protein encodes a signal peptide. The protein has approximately 50% homology with both the prostatic and the lysosomal acid phosphatases, and the position of the cysteine residues, the N-glycosylation sites, and the histidine catalytic site are conserved among the three proteins. The testicular acid phosphatase gene is up-regulated by androgens and is down-regulated by estrogens in the prostate cancer cell line LNCaP. Our preliminary results indicate that this gene exhibits a lower level of expression in testicular cancer tissues than in their normal counterparts.     

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.     

Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions

The effect of ferulic acid was studied on γ-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body γ-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of ferulic acid 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensuration with DNA repair. Administration of 50 mg/kg body weight of ferulic acid after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in ferulic acid treated animals. (Mol Cell Biochem xxx: 209–217, 2005)     

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.     

Cloning of human myeloid-associated differentiation marker (MYADM) gene whose expression was up-regulated in NB4 cells induced by all-trans retinoic acid

A full-length cDNA of 3192 bp isolated from human bone marrow cDNA library was predicted an ORF encoding 298 amino acids. The deduced protein, containing seven putative transmembrane segments and sharing 75.8% amino acid identity with mouse Myadm protein, was named as human MYADM. The results of Northern blot analysis showed that MYADM was ubiquitously expressed in 15 of 16 adult tissues tested, except thymus. To determine whether the novel human gene was involved in hematopoietic differentiation process as mouse Myadm did, we examined the mRNA expressive abundance of this gene between normal bone marrow cells and peripheral blood leukocytes, and detected the expression change in NB4 cells induced by all–trans retinoic acid at different induce time by the semi-quantitative RT-PCR. The results showed that the expression of the novel gene was not only significantly higher in peripheral blood leukocytes than in bone marrow cells, but also significantly up-regulated when the NB4 cells(derived from a patient with acute promyelocytic leukemia) were induced by all-trans retinoic acid (ATRA) for 48hr. It is suggested that human MYADM was also associated with the differentiation of hematopoietic cells or acute promyelocytic leukemia cells. In addition, MYADM was mapped to human chromosome 19q13.33-q13.4 by Radiation Hybrid mapping, and it consists of 3 exons and 2 introns and spans a 7.1-Kb genomic region.     

Native blot and immunotransfer of human prostatic acid phosphatase isozymes

Agarose gel isoelectrofocusing is used to separate the isozymes of human prostatic acid phosphatase with retention of enzyme activity. The native blotting of the isozymes onto a nitrocellulose membrane increases the sensitivity of the enzyme stain and is suitable for analysis of isozymes in prostate tissue, which contains little nonprostatic acid phosphatase. The specificity of the transfer is increased by treating the membrane with antibody to human prostatic acid phosphatase prior to the transfer. The specificity of the antibody is conferred to the membrane resulting in a transfer specific for prostatic acid phosphatase. The immunotransfer procedure is applicable to serum which contains appreciable amounts of nonprostatic acid phosphatase.     

Prostatic acid phosphatase, a neglected ectonucleotidase

Two recent papers reveal that the soluble and secreted prostatic acid phosphatase, an enzyme that has long served as a diagnostic marker for prostate cancer, has a membrane-bound splice variant. This enzyme exhibits ecto-5′-nucleotidase activity, is widely distributed, and implicated in the formation of chronic pain. While prostatic acid phosphatase hydrolyzes phosphomonoesters other than 5′-nucleoside monophosphates these novel data suggest that, in addition to ecto-5′-nucleotidase and the alkaline phosphatases, prostatic acid phosphatase must be taken into account in future studies on extracellular adenosine production.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

Upregulation of endothelin 1 and its precursor by IL-1beta, TNF-alpha, and TGF-beta in the PC3 human prostate cancer cell line

Increasing evidence indicates that endothelin 1 (ET-1) is implicated in prostate tumour progression. However, data on ET-1 regulation in human prostate and prostate cancer cell lines are lacking. In this study, regulation of ET-1 and its precursor big ET-1, using PC3 cells, a human bone metastatic prostatic carcinoma cell line, was addressed. ET-1 and big ET-1 assays demonstrated greater secretion of both peptides in the presence of 10% fetal calf serum (FCS) as compared with 0.5% FCS. Incubation of PC3 cells in the absence and presence of various cytokines and growth factors known to be implicated in prostate stroma-epithelium interactions, revealed that IL-6, FGF7/KGF and FGF2/bFGF had no effect on ET-1 and big ET-1 secretion, whereas interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) stimulated their secretion in a concentration-dependent manner. Binding experiments indicated the presence of specific ET-1 receptors in PC3 cells: Kdapp = 1.1 x 0.2 x 10(-10)M, Bmax = 2660 +/- 390 sites/cell. Data analysis demonstrated the presence of only the ETA receptor subtype in PC3 cells. In conclusion, our results indicate that the implication of ET-1 in prostate cancer is likely to be mediated via paracrine/autocrine control of cell factors.