Possible involvement of prolactin in endocrine-resistant metastatic prostate cancer.

The hormone resistance of prostate cancer has been proved to depend at least in part on enhanced neuroendocrine activity and the resultant increase in blood concentrations of chromogranin A. Other experimental observations have suggested the involvement of prolactin (PRL), which appears to be a potential growth factor for prostate cancer. Abnormally high levels of PRL have been detected in metastatic prostate cancer, but the clinical significance of this finding has still to be clarified. In an attempt to explain the prognostic significance of serum PRL levels in prostate cancer, in this preliminary study we have analyzed the PRL levels in a group of metastatic prostate cancer patients with hormone-dependent or hormone-resistant cancer. The study included 50 patients with metastatic prostate cancer, 15 of whom had hormone-resistant tumors. The serum levels of PRL were measured by the RIA method. Abnormally high concentrations of PRL were found in 11/50 (22%) patients. Moreover, the percent of patients with cancer-related hyperprolactinemia was significantly higher in the hormone-resistant group than in the hormone-dependent group (8/15 vs 3/35, p < 0.01). This study confirms the possible existence of a hyperprolactinemic state in metastatic prostate cancer, as previously reported by other authors. Moreover, it appears to demonstrate that the occurrence of hyperprolactinemia is more frequent in hormone-resistant neoplasms, suggesting the possible involvement of PRL in hormone independence. Further studies concomitantly evaluating PRL and chromogranin A blood concentrations will be necessary to establish whether the hyperprolactinemia precedes and promotes the onset of hormone resistance in prostate cancer, or whether it is simply a consequence of the hormone independence.

The favorable prognostic significance of surgery-induced hyperprolactinemia in node-positive breast cancer patients: ten-year disease-free survival results.

It has been shown that each manipulation of the mammary region, including breast surgery, may stimulate prolactin secretion. However, it has also been observed that in more than 50% of breast cancer patients surgical removal of the tumor is not followed by enhanced prolactin secretion. This might be indicative of an altered psychoneuroendocrine control of the mammary gland, which could lead to the onset of more biologically aggressive breast cancer. In fact, surgery-induced hyperprolactinemia has been proven to be associated with a better prognosis in terms of survival in node-negative breast cancer patients. The present study was performed to investigate the impact of postoperative hyperprolactinemia on the disease-free survival (DFS) of breast cancer patients with axillary node involvement. The study included 100 consecutive node-positive breast cancer patients who were followed for at least 10 years. Surgery-induced hyperprolactinemia occurred in 45/100 (45%) patients without any significant correlation with the main prognostic variables including number of involved nodes and ER status. The two groups of patients received the same adjuvant therapies. After a median follow-up of 151 months, the recurrence rate in patients with surgery-induced hyperprolactinemia was significantly lower than in patients with no postoperative hyperprolactinemia (23/45 vs 43/55, p<0.01). Moreover, DFS was significantly longer in hyperprolactinemic patients than in patients who had no enhanced secretion of prolactin postoperatively. In agreement with the results described previously in node-negative breast cancer, our study demonstrates the favorable prognostic significance of surgery-induced hyperprolactinemia in terms of DFS duration also in breast cancer patients with axillary node involvement, independent of the other well-known prognostic variables, thereby confirming that the psychoneuroendocrine status of cancer patients may influence the prognosis of their disease.

Possible involvement of prolactin in endocrine-resistant metastatic prostate cancer.

The hormone resistance of prostate cancer has been proved to depend at least in part on enhanced neuroendocrine activity and the resultant increase in blood concentrations of chromogranin A. Other experimental observations have suggested the involvement of prolactin (PRL), which appears to be a potential growth factor for prostate cancer. Abnormally high levels of PRL have been detected in metastatic prostate cancer, but the clinical significance of this finding has still to be clarified. In an attempt to explain the prognostic significance of serum PRL levels in prostate cancer, in this preliminary study we have analyzed the PRL levels in a group of metastatic prostate cancer patients with hormone-dependent or hormone-resistant cancer. The study included 50 patients with metastatic prostate cancer, 15 of whom had hormone-resistant tumors. The serum levels of PRL were measured by the RIA method. Abnormally high concentrations of PRL were found in 11/50 (22%) patients. Moreover, the percent of patients with cancer-related hyperprolactinemia was significantly higher in the hormone-resistant group than in the hormone-dependent group (8/15 vs 3/35, p<0.01). This study confirms the possible existence of a hyperprolactinemic state in metastatic prostate cancer, as previously reported by other authors. Moreover, it appears to demonstrate that the occurrence of hyperprolactinemia is more frequent in hormone-resistant neoplasms, suggesting the possible involvement of PRL in hormone independence. Further studies concomitantly evaluating PRL and chromogranin A blood concentrations will be necessary to establish whether the hyperprolactinemia precedes and promotes the onset of hormone resistance in prostate cancer, or whether it is simply a consequence of the hormone independence. (Int J Biol Markers 2005; 20: 123-5).

Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?

Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.

On the existence of a cartilage-like proteoglycan and link proteins in the central nervous system.

Monoclonal antibodies (mAbs) against the major constituents of cartilage extracellular matrix, aggrecan and link protein, were screened by indirect immunofluorescence on frozen sections of bovine spinal cord. Antibodies against aggrecan and link protein gave rise to very similar perineuronal labeling in spinal cord gray matter. Aggrecan and link protein reactivities were seen in other regions of the central nervous system (CNS), although their distributions were not always coincident. Pretreatment of the tissue section with Streptomyces hyaluronidase, which is hyaluronate-specific, led to the loss of both reactivities. On Western blots, anti-aggrecan mAbs reacted with a large chondroitin sulfate proteoglycan. The chondroitinase-treated CNS proteoglycan co-migrated with the chondroitinase- and keratanase-treated cartilage proteoglycan. In CNS tissue homogenates, the addition of Streptomyces hyaluronidase brought about the release of the proteoglycan from the tissue. Anti-link protein mAbs were reactive with two species in the bovine CNS, the mobilities of which were very similar to those of the cartilage link proteins. The release of these species from the tissue required hyaluronidase. A rabbit antiserum against aggrecan was used to identify a similar proteoglycan in the rat CNS. In spinal cord-derived cell cultures, the labeled material was associated with astrocytes. An aggrecan cDNA hybridized to a 9.5 kb mRNA in the rat CNS. We conclude that the perineuronal matrix consists, in part, of a hyaluronate-bound aggrecan-like proteoglycan and link proteins, and that the former is produced by astrocytes.

An attempt to control the polychlorocatechol pigment production during 3-chlorobenzoate aerobic co-metabolism in growing-cell batch culture.

A strain of Pseudomonas fluorescens, designed CP30, was capable of co-metabolizing 3-chlorobenzoic acid via chlorocatechols, thus producing a polychlorocatechol black pigment. The pigment production was found to be enzyme-mediated in P. fluorescens cultures; however, the results of the autoxidation studies suggested that a large amount of pigment could derive from the simple autoxidation of the chlorocatechols accumulated in the CP30 culture during the 3-chlorobenzoate degradation. The polymerization of chlorocatechols should reduce the toxicity of the culture and generally resulted in a large production of a water-insoluble (i.e. a easily removable) pigment with the release a large amount of chloride ions. Both 3-chlorobenzoate degradation and pigment production were stimulated and inhibited in the same way by high and low pH values, respectively, in CP30 cultures; the culture conditions which permitted an extensive 3-chlorobenzoate degradation with the lowest pigment production were not identified. However, on the basis of the detoxification property of the pigment production process, high pH and dissolved oxygen concentrations appears to be the most suitable culture conditions for an enhanced detoxicant treatment of 3-clorobenzoate-contaminated waste water.

A role for extracellular matrix degradation and matrix metalloproteinases in senile dementia?

In brain as in cartilage, the extracellular matrix contains aggregates formed by hyaluronic acid (HA) and proteoglycans. In osteoarthritic cartilage, release of the proteoglycans from the aggregates by cleavage of the HA-binding region results in the accumulation of the HA-binding region and in the fragmentation of the released proteoglycans. Stromelysin, a matrix neutral metalloproteinase, is one of the enzymes responsible for the cleavage of the HA-binding region. We suggest that a similar process also occurs in senile dementia. The brain proteoglycan contains sequences identical to those of aggrecan, which are recognized and cleaved by stromelysin, and is, in fact, susceptible to stromelysin digestion. Monoclonal antibodies reacting with glial HA-binding protein, but not with the parent protein, stained several senile plaques as defined by their reactivity with antibodies to the amyloid-beta protein in double-labeling experiments.

Colocalization of tenascin with versican, a hyaluronate-binding chondroitin sulfate proteoglycan.

Rabbit antisera against tenascin, a large extracellular matrix protein, in conjunction with monoclonal antibodies of mouse origin against versican, a large hyaluronate-binding proteoglycan, were used to make a comparative study of the distribution of the two antigens in the same cryostat sections by double immunofluorescence. In the central nervous system, tenascin was invariably associated with versican, but the reverse was not true, in that versican was also found where tenascin was not detectable, particularly in gray matter. There were major species differences in the distribution of tenascin in the central nervous system. In the cow, tenascin was found in cerebral and spinal cord white matter and in the granule cell layer of the cerebellum. In the human brain, tenascin was found in cerebral white matter but not in the cerebellum. In the rat, tenascin was mainly confined to brain periventricular layer and spinal cord white matter. During the development of the cerebellum of the rat, the tenascin immunoreactivity decreased, and a lower molecular weight band appeared (J1-160/180/restrictin?) and persisted throughout adulthood. Tenascin expression was a relatively late event in the development of the rat central nervous system, immunoreactivity being first observed after birth. In the rat embryo, tenascin was found to co-localize with versican in precartilaginous mesenchyme and in connective tissue underlying epithelia. The colocalization of versican with tenascin suggests that versican may be the tenascin (cytotactin)-associated proteoglycan reported in the literature.

Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix.

Hyaluronic acid (HA) plays the main structural role in the formation of brain extracellular matrix (ECM). The extracellular space appears empty by electron microscopy because HA is readily dissolved during the preparation of tissues for ultrastructural studies. The HA-binding proteins so far identified in brain ECM are versican, aggrecan and the glial HA-binding protein. Versican is a large fibroblast proteoglycan preferentially expressed in embryonic cartilage at the time of mesenchymal condensation. Glial HA-binding protein (GHAP) is probably a proteolytic product of versican corresponding to its HA-binding amino-terminal domain. It is mainly a white-matter protein, suggesting that the proteinase responsible for its cleavage from versican is normally activated in this location. Versican is found in both white matter and gray matter, where it forms pericellular coats around large neurons. Aggrecan, the aggregating proteoglycan of mature cartilage, co-localizes with versican in this location. In white matter, the localization of GHAP and versican is identical to that of the glial fibrillary acid protein, suggesting that both proteins are produced by astrocytes. An important difference between GHAP and versican is that GHAP but not versican is released from the tissues by hyaluronidase digestion, which suggests that versican is anchored to the cell membranes lining the extracellular space. GHAP was localized at the ultrastructural level in the granule cell layer of rat cerebellum, the only region of gray matter that is positive for GHAP in this species. Rats were perfused with aqueous fixatives containing cetylpyridinium chloride or tannic acid to prevent the solubilization of HA. GHAP is found throughout the extracellular space, the synaptic clefts being a notable exception. GHAP appears late in development, and the same is true for versican, the characteristic perineuronal coats first becoming apparent in the third postnatal week. It is suggested that a marked change occurs in the structure of brain ECM when HA-binding proteins first appear, and that the change is similar to that observed in prechondrogenic mesenchyme, i.e., reduction of the extracellular space and cell aggregation.

Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration.

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate-binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2-3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2-3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase-injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase-injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500 microns) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline-injected controls.

Versican, a hyaluronate-binding proteoglycan of embryonal precartilaginous mesenchyma, is mainly expressed postnatally in rat brain.

The localization of versican, a large hyaluronate-binding fibroblast proteoglycan, was studied in rat prenatal and postnatal development. In adult rat white matter and cerebellum, the distribution of versican was identical to that previously reported for brain-specific glial hyaluronate-binding protein (GHAP). Versican was also found in gray matter where it formed characteristic coats around large neurons. It was also found in peripheral tissues, namely, kidney medulla, myotendinous junctions, and endoneurial and endomysial sheaths. In rat embryo the most notable finding was the presence of large amounts of versican immunoreactive material in precartilaginous mesenchyma. In embryonal CNS, versican was mainly confined to the marginal zone on the surface of the cerebral hemispheres. Versican expression mainly occurred postnatally in brain and spinal cord. In spinal cord white matter, versican immunoreactivity was already present in 3-day-old rats and preceded the appearance of GHAP, which was first detected on day 13 after the onset of myelination. Versican expression was markedly delayed in gray matter. The characteristic perineuronal coats were first observed on day 21 in the cerebral cortex. It is concluded that, with the exception of hyaluronate, brain extracellular matrix (ECM) is mainly produced postnatally and that the ECM protein produced by brain cells, most likely astrocytes, is similar to that produced by precartilaginous mesenchyma.

Hyaluronate binding and CD44 expression in human glioblastoma cells and astrocytes.

CD44 is an integral membrane glycoprotein of approximately 90 kDa which has been implicated in the binding of hyaluronate to the cell surface. The expression of CD44 in astrocytes was investigated by means of indirect immunofluorescence on cultured cells. The vast majority of these cells were found to express CD44. Western blot analysis of these cells revealed a highly polydisperse species having an M(r) corresponding to 74-86 kDa. In order to visualize hyaluronate-binding cells, living cultures were probed with fluorescein-conjugated hyaluronate (FI-HA). Some astrocytes were able to bind FI-HA, provided that they were first treated with hyaluronidase. Streptomyces hyaluronidase, which is hyaluronate-specific, was effective in exposing the hyaluronate-binding capacity of these cells. This leads one to conclude that hyaluronate is bound to the surface of these cells and that it masks their capacity to bind hyaluronate. Provided that they were first treated with hyaluronidase, the U-87 MG (glioblastoma-astrocytoma), U-373 MG (glioblastoma), and Hs 683 (glioma) cell lines were also able to bind FI-HA. The U-138 MG (glioblastoma) cell line was unable to bind FI-HA, with or without prior hyaluronidase treatment. A quantitative assay was developed with the use of [3H]hyaluronate ([3H]HA). This revealed the binding to be highly specific, inasmuch as the addition of unlabeled hyaluronate, but not other glycosaminoglycans, was effective in inhibiting the binding of the [3H]HA. An anti-CD44 monoclonal antibody, 50B4, was able to inhibit the binding of the [3H]HA to the U-373 MG cell line. In this cell line, then, CD44 functions as a hyaluronate receptor and one may infer that this is also the case in some astrocytes.

Isolation of a large aggregating proteoglycan from human brain.

A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.

The extracellular matrix of cerebral gray matter: Golgi's pericellular net and Nissl's nervösen grau revisited.

Glial hyaluronate-binding protein (GHAP) and a large aggregating chondroitin sulfate proteoglycan (Ag-Pg) similar to a fibroblast proteoglycan (versican) were localized in bovine, dog and cat central nervous system (CNS) gray matter by indirect immunofluorescence. The distribution of the two hyaluronate-binding proteins was identical with that of hyaluronate, an extracellular glycosaminoglycan. All substances formed a finely reticulated mesh in the neuropil with a condensation of the stain around large neurons. It is concluded that in gray matter, as in white matter, the extracellular matrix (ECM) contains hyaluronate-protein aggregates. We suggest that the hyaluronate-protein aggregates correspond to the pericellular network first described by Golgi.

The astrocyte--extracellular matrix complex in CNS myelinated tracts: a comparative study on the distribution of hyaluronate in rat, goldfish and lamprey.

The localization of hyaluronate was studied in the CNS of rat, goldfish and lamprey. Cryostat sections were incubated with glial hyaluronate-binding protein of human origin and stained by indirect immunofluorescence with glial hyaluronate binding protein antibodies not reaching with rat and fish. As previously reported for glial hyaluronate-binding protein and glial fibrillary acidic protein, hyaluronate and glial fibrillary acidic protein had a similar distribution in rat spinal cord and optic nerve, both substances forming ring-like structures around individual myelinated axons. A similar periaxonal distribution was observed in goldfish spinal cord and medulla, except that the rings were much wider, to accommodate the large goldfish axons. The glial fibrillary acidic protein-positive neuroglial tissue forming distinctive structures in goldfish vagal lobes also stained for hyaluronate. In both rat and goldfish spinal cord, motoneurons were surrounded by a hyaluronate coat. Goldfish optic nerve and lamprey spinal cord were hyaluronate-negative and, as previously reported, they stained for keratin but not for glial fibrillary acidic protein. The findings suggest that hyaluronate in CNS fibre tracts in a product of glial fibrillary acidic protein-positive neuroglia. They also suggest that the appearance of glial fibrillary acidic protein-positive neuroglia and the formation of a hyaluronate-bound extracellular matrix are related phenomena in phylogeny.

The extracellular matrix of rat spinal cord: a comparative study on the localization of hyaluronic acid, glial hyaluronate-binding protein, and chondroitin sulfate proteoglycan.

The localization of hyaluronic acid (HA), glial hyaluronate-binding protein (GHAP), and chondroitin sulfate (CS) proteoglycan was compared in cryostat sections of rat spinal cord. HA, GHAP, and CS proteoglycan were similarly distributed in white matter where they surrounded myelinated axons. In gray matter, large motoneurons were surrounded by a rim of reaction product in sections stained for HA and CS proteoglycan. GHAP immunoreactivity as well as HA had disappeared in hyaluronidase-digested sections, while CS proteoglycan immunoreactivity was not abolished under these conditions.

Co-localization of hyaluronic acid and chondroitin sulfate proteoglycan in rat cerebral cortex.

The distribution of hyaluronate (HA) and chondroitin sulfate (CS) proteoglycan in the rat cerebral cortex was compared. For the localization of HA, the sections were incubated with human glial hyaluronate-binding protein (GHAP) and then reacted with monoclonal or polyclonal antibodies to GHAP. Polyclonal antibodies raised in rabbit were used for double-labeling experiments with monoclonal antibodies raised in mice and reacting with CS proteoglycans. Little reactivity was observed in rat cerebral cortex with polyclonal GHAP antibodies if the sections were not incubated with GHAP. Monoclonal antibodies to GHAP did not react with murine tissues. CS proteoglycans were localized in chondroitinase-digested sections with monoclonal antibodies reacting with the 4-sulfated oligosaccharide stubs formed by the digestion with chondroitinase ABC of CS side chains. In the rat cerebral cortex, the distribution of CS proteoglycans was similar to that reported by Bertolotto, A., Rocca, G. and Schiffer, D., J. Neurol. Sci., 100 (1990) 113-123, and his collaborators using the same antibodies. Many neurons mainly located in the upper and deep cortical layers were surrounded by CS immunoreactive material. Several (but not all) CS-positive neurons also stained for HA with an identical distribution except that in most instances the staining was confined to the periphery of the perikaryon and did not extend to the dendritic tree. The finding suggests that cerebral cortex CS proteoglycan is capable of interacting with HA.

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.