Expression of cell cycle-regulated proteins in prostate cancer.

Markers of cellular proliferation have proven to be useful prognostic markers in several tumor types. Recently, immunoreactivity for cyclins was found to provide an independent marker of tumor proliferation in breast cancer. In this study, we sought to determine the pattern of immunoreactivity for cyclins A, B, E, and Ki-67 in surgically resected prostate cancer and to determine their possible prognostic significance. Twenty-eight tumors of American Urological Association stages B and C were selected for study. Immunoreactivity for cyclins A and B was detected in most tumors and was present at significantly reduced levels as compared with breast cancer. Staining for cyclin E was present in four tumors and was present only in focal areas in two of the four. Such focal variation in expression of cell cycle regulators may reflect genetic instability in a tumor. Immunoreactivity for cyclins A and B was correlated with both Ki-67 index (the percentage of cells with Ki-67 immunoreactivity) and with each other. A Ki-67 index greater than 4.0 was associated with shorter time to prostate-specific antigen-detected relapse (P = 0.026). The fraction of cells staining for cyclins A and B divided by the fraction of cells staining for Ki-67 [(A+B)/K] was highly predictive of relapse, with values less than 0.50 associated with more rapid progression (P < 0.001). This latter result remained statistically significant after controlling for Gleason score by stratification. Our results suggest that immunoreactivity for markers of cellular proliferation may provide useful prognostic information in localized prostate cancer, and they need to be validated in a larger numbers of patients.

Practical methods of mutation detection.

The past few years have seen a marked increase in the demand for practical methods of mutation detection. Over this period of time, both the development of new methods and improvements in existing methods have made mutation detection in the research setting a less cumbersome task. Nevertheless, all of the commonly used approaches to this problem are labor intensive, and truly practical mutation detection methods in the clinical setting will depend on the development of more fully automated technology.

Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases.

We have explored the application of the bacteriophage resolvases T4 endonuclease VII and T7 endonuclease I for detecting mutations in genomic DNA. Heteroduplex DNA fragments prepared by amplification from DNA containing known mutations were cleaved by one or both enzymes at nucleotide mismatches created by 3 of 3 short deletions and 13 of 14 point mutations in fragments as large as 940 basepairs. Heteroduplexes representing all four classes of possible single nucleotide mismatches were cleaved, and the sizes of the cleavage products generated correlated with the location of the mutation. We conclude that bacteriophage resolvases may be useful reagents for the rapid screening of DNA for mutations.

Analysis of androgen receptor DNA reveals the independent clonal origins of uterine leiomyomata and the secondary nature of cytogenetic aberrations in the development of leiomyomata.

Uterine leiomyomata are thought to be monoclonal neoplasms. Accordingly, investigations of clonality with G6PD isoforms used as a marker for X chromosome inactivation have suggested independent origins for multiple tumors within individual uteri. However, results from a recent study assessing methylation differences between DNA of active and inactive X chromosomes have been interpreted to suggest that multiple tumors may arise from a common precursor. We have examined the clonality of 36 leiomyomata from 16 patients by analyzing X chromosome inactivation as indicated by the methylation status of the X-linked androgen receptor gene. As shown by this assay, all informative leiomyomata were monoclonal in origin. In patients with multiple leiomyomata, a random distribution of inactivation between the X homologs was noted, consistent with an independent origin of each tumor. Cytogenetic analysis was also performed on short-term cell cultures of 27 of the 36 tumors. In each of two tumors that had both cells with a clonal karyotypic abnormality and karyotypically normal cells, DNA prepared from short-term cultures showed a monoclonal pattern of X inactivation identical to that of the leiomyoma from which they were derived. These data suggest that karyotypically normal cells present in short-term cultures of uterine leiomyomata are part of the tumor clone, and that clonal expansion of tumor cells precedes the development of cytogenetic aberrations.

Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.

Analysis of clonality by X chromosome inactivation has proven to be a powerful strategy in the study of neoplastic and preneoplastic disorders (P. J. Fialkow, Biochim. Biophys. Acta, 458: 283-321, 1976; B. Vogelstein et al., Cancer Res., 47: 4806-4813, 1987). Recently, the gene for the androgen receptor has been shown to be a highly polymorphic locus in which methylation of DNA correlates with inactivation of one or the other X homologue (R. C. Allen et al., Am. J. Hum. Genet., 51: 1229-1239, 1992). Unlike other loci used for analysis of X inactivation, the methylation sites within the androgen receptor gene lie close to the region of DNA containing the polymorphism. Consequently, it should be possible to use methylation-sensitive restriction enzymes and polymerase chain reaction to study differential methylation among alleles of this gene in formalin-fixed and paraffin-embedded archival tissue specimens. To investigate this question, we performed clonal analysis on a variety of randomly selected, formalin-fixed, paraffin-embedded tumor tissues from 15 women. Thirteen of the women were found to be heterozygous for polymorphisms at the androgen receptor locus. Among these women, 11 tumors were clearly clonal in assays of methylation of the androgen receptor gene. Interpretation of results for the remaining two cases was complicated because of a skewed pattern of X chromosome inactivation found in normal control tissues. We conclude that analysis of methylation in the androgen receptor gene should allow study of clonality in most formalin-fixed, paraffin-embedded tissue specimens from women, including small preneoplastic lesions or rare conditions for which sufficient material is not available for analysis by other techniques.

[3H]R05-4864 and [3H]flunitrazepam binding in kainate-lesioned rat striatum and in temporal cortex of brains from patients with senile dementia of the Alzheimer type.

In agreement with other workers we report increased [3H]R05-4864 binding in kainate-lesioned rat striatum. [3H]R05-4864 binding, a possible glial marker, was also increased in temporal cortex obtained post-mortem from patients with Alzheimer's disease. [3H]Flunitrazepam binding was decreased in these brain samples, possibly indicative of neuronal cell loss. It is suggested that the poor binding characteristics of [3H]R05-4864 in human brain samples may limit its usefulness in assessing gliosis.

Preferential inhibition of ligand binding to calf striatal dopamine D1 receptors by SCH 23390.

The phenylbenzazepine derivative, SCH 23390 was found to be a potent inhibitor of 3H-piflutixol binding to calf striatal dopamine D1 receptors. In contrast SCH 23390 was only a weak inhibitor of 3H-spiperone binding to dopamine D2 receptors. Although possessing some activity at serotonin S2 and alpha 2 adrenergic receptors, SCH 23390 appears to be a potent and selective D1 receptor antagonist.

The effects of cholecystokinin on dopaminergic mechanisms in rat striatum.

Intraventricular administration of cholecystokinin-octapeptide in unanesthetized rats results in a significant reduction in the number of [3H]spiperone binding sites. Striatal levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly reduced, whereas there was no significant reduction in the levels of the serotonin metabolite 5-hydroxyindoleacetic acid.

Benzodiazepine receptors: preferential stimulation of Type 1 receptors by pentobarbital.

The binding of [3H]flunitrazepam (FLU) to Type 1 and Type 2 benzodiazepine receptors in rat cerebellum and cerebral cortex was differentiated by the addition of 200 nM CL218,872 which preferentially displaces [3H]FLU from Type 1 receptors. Type 1 but not Type 2 receptor binding was significantly stimulated by 1 mM sodium pentobarbital.

77Br-p-bromospiperone: a ligand for in vivo labelling of dopamine receptors.

A high striatum: cerebellum ratio of 77Br-p-bromospiperone (77Br-BrSp) was observed in rat brain following tail vein injection of the drug. Striatal 77Br-BrSp was stereospecifically displaced by the isomers of flupenthixol. After chronic haloperidol administration striatal dopamine receptor supersensitivity was demonstrated both by increased 3H-spiperone binding to striatal membranes in vitro and by increased striatal 77Br-BrSp content. These results confirm and extend previous findings and enhance interest in the use of 77Br-BrSp for the in vivo assessment of central dopamine receptors in man.

[3H]etorphine binding in rat striatum following lesions of the raphe nuclei.

Following 5,7-dihydroxytryptamine lesions of the rat raphe nuclei a significant reduction in striatal serotonin concentrations was observed. However, with [3H]etorphine as ligand no change in opiate receptor binding was observed in striatal membranes. Moreover, when [3H]etorphine binding was resolved into its mu- and delta + kappa-components no change in either component was observed compared with controls. It is concluded that opiate receptors are not on striatal serotonergic nerve terminals whose cell bodies are located in the raphe nuclei.

Binding of a radiolabeled triazolopyridazine to a subtype of benzodiazepine receptor in the rat cerebellum.

The triazolopyridazine (TPZ) drugs, typified by CL218,872 (CL), have a relatively higher affinity for a subpopulation of benzodiazepine (BZ) receptors. The binding of radiolabeled CL to membranes from rat cerebellum, a region enriched in the TPZ-preferring ("Type 1") BZ receptor, was characterized and compared with that of [3H]flunitrazepam ([3H]FLU) in the same preparation. [3H]CL had clonazepam displaceable binding which was saturable. The Kd was approximately 21 nM and the Bmax was approximately 600 fmol/mg of protein. [3H]CL binding was similar to that for [3H]FLU in that exogenous gamma-aminobutyric acid (GABA) enhanced the binding; however, [3H]CL binding differed from that for [3H]FLU in that anions, cartazolate and pentobarbital did not enhance [3H]CL binding. These data suggest that [3H]CL binds to the Type 1 BZ receptor in a manner different from that of a BZ drug such as FLU. Inasmuch as GABA enhances [3H]CL binding, but anions, cartazolate and pentobarbital do not, [3H]CL may bind to the Type 1 BZ receptor in such a way that it interacts with the GABA site, but perhaps not directly with the ionophore or the postulated pyrazolopyridine-barbiturate site. Thus, TPZ drugs may affect the GABA receptor complex in a different or perhaps less extensive way than the BZs. This, in addition to the regional localization of the Type 1 receptor, may be an important part of the mechanism of action of the TPZs.

Immunofluorescent staining of rat brain glial cells with multiple sclerosis serum.

Antibodies directed against glial cells may be involved in autoimmunity in multiple sclerosis (MS). Using a tissue culture system, the presence of glial cell antibodies in MS-patient serum was detected through immunofluorescent technique. Thirty one of 73 MS-sera were strongly positive for anti-glial cells, 13 were equivocal and 29 were negative. The antibody staining was either cytoplasmic or associated with cell surface membrane, and involved IgG type of antibody.