Cytoskeletal organization and cell motility correlates with metastatic potential and state of differentiation in prostate cancer.

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.

Novel antineoplastic isochalcones inhibit the expression of cyclooxygenase 1,2 and EGF in human prostate cancer cell line LNCaP.

Experiments were conducted to determine the effects of novel anti-neoplastic isochalcones (DJ compounds), on cyclooxyegenase 1 and 2 (COX-1 and COX-2) enzyme expression in androgen receptor dependent human prostate cancer cell line LNCaP. Results from Western blot analysis and cell flow cytometry showed that DJ52 and DJ53 decreased the steady state levels of COX-1 and COX-2 protein levels in a dose dependent manner. In addition, DJ52 and DJ53 decreased the levels of epidermal growth factor (EGF) in LNCaP cells. In this study, we report that novel isochalcones decreased COX-1, COX-2 and EGF levels as well as LNCaP cellular growth in a dose responsive manner. Our findings indicate that relative decreases in COX-1, COX-2 and EGF expressions might serve as indicators of tumor growth inhibition in prostate neoplasms.

Safety and effect on anti-Chlamydia pneumoniae antibody titres of a 1 month course of daily azithromycin in adults with coronary artery disease.

A pilot study of azithromycin treatment following percutaneous coronary revascularization procedures was performed to assess safety and the effect of azithromycin treatment on anti-Chlamydia pneumoniae antibody titres. Patients were randomized to a 1 month course of azithromycin (total dose of 8.0 g) or placebo. Safety and compliance were assessed at 2 and 4 weeks and serological testing was performed on samples obtained at enrolment and at 6 months post-enrolment. Azithromycin was well tolerated at this dose. No effect of treatment on antibody titres was demonstrated. These results support further clinical trials to assess the effect of azithromycin treatment on cardiovascular disease outcomes.

Invasive potential and substrate dependence of attachment in the dunning R-3327 rat prostate adenocarcinoma model.

Cancer cell attachment to and invasion of the extracellular matrix has been associated with the metastatic potential of cell lines of the Dunning R-3327 rat prostatic adenocarcinoma model. We investigated the cell-matrix interactions of prostate tumor cells by comparing the invasive ability through reconstructed extracellular matrix and attachment upon EHS NATRIX (natural extracellular matrix), fibronectin, laminin, and collagen Type IV. We observed a correlation between metastatic potential and substrate dependence of attachment in prostate cancer cells. Nonmetastatic AT-1 cells possessed a higher adhesive potential to extracellular matrix components than the highly metastatic cells (ML, MLL and AT-3). It was also found that the invasive potential of the three highly metastatic cell lines was significantly higher than that of the nonmetastatic cell line. Here, it is reported that the ability to traverse a matrigel matrix correlates with their metastatic potential. These observations suggest that the extracellular matrix components are highly involved in influencing prostate cancer cell activities. In addition, we investigated the effects of two differentiation agents, retinoic acid (RA) and difluoromethylornithine (DFMO), on the adhesive and invasive profiles of the tumor cells. After treatment with both agents, adhesion was increased to levels not different from nonmetastatic cells. Furthermore, the ability of highly metastatic cells to traverse a matrigel barrier was significantly reduced after treatment with both differentiation agents. These results suggest that RA and DFMO are capable in reversing the metastatic potential of prostate cancer cells in vitro and may give a possible insight into their role as potential therapeutic agents in vivo.

Genomic methylation patterns of the Dunning R-3327 prostate adenocarcinoma system.

Genomic methylation patterns of Dunning R-3327 cell lines anaplastic tumor-1 (AT-1), anaplastic tumor-3 (AT-3), metastasis-lymph and lung (Mat-LyLu) and metastasis-lung (Mat-Lu), and Mat-LyLu cells treated with difluoromethylornithine (DFMO), and retinoic acid (RA) have been analyzed. Each cell line was digested with HpaII and MspI restriction endonuclease enzymes to characterize methylation patterns, at the interior cytosine of the sequence CmCGG. Identical molecular weight banding patterns were found for both HpaII and MspI digests in normal dorsal prostate (NDP) used as a control. Both the treated and non-treated Dunning R-3327 cells digested with HpaII and MspI, displayed similar banding profiles from those seen in NDP solid tissues, indicative of a progressive loss of methylation at CCGG sites.

Point mutations in the Ki-ras2 gene of codon 12 in the Dunning R-3327 Prostatic Adenocarcinoma system.

Detection of point mutations in the Dunning System with the Ki-ras2 gene in the first and second positions of codon 12 exon I, has been performed using allele-specific oligonucleotide (ASO) primers. PCR generated 94bp templates and genomic DNA extracted from the Dunning cell lines AT-1, AT-3, Mat-Lu, and Mat-Lylu were tested. Our investigation revealed that the first and second positions of codon 12 have undergone either transitions or transversions from the wild type sequence (GG). Dorsal prostate (DP) solid tissues used as controls revealed the wild type configuration as well as transversions at both positions, in addition to a transition in the first position for Mat-Lu. The most aberrant of the Dunning lines (AT-3 and Mat-LyLu) collectively displayed sequence changes (transitions and transversions) with no evidence of the wild type configuration. These findings are supportive of biometric studies (area, shape factor, and motility) along with adhesion and invasion assays done in our laboratory in correlating genotypic and phenotypic properties to metastatic potential.

Biometric assessment of prostate cancer's metastatic potential.

Currently, no protocol exists that can assess the metastatic potential of prostate adenocarcinoma. The reason for this is partly due to the lack of information on cellular changes that result in a tumor cell's becoming metastatic. In this investigation, attempts were made to devise a method that correlated with the metastatic potential of AT-1, Mat-Lu, and Mat-LyLu cell lines of the Dunning R-3327 rat prostatic adenocarcinoma system. To accomplish this, we applied BioQuant biometric parameters, i.e., area, shape factor, and cell motility. AT-1 had a lower shape factor and a greater area as compared with the more highly metastatic Mat-Lu subline. No significant difference in area or shape factor was detected between the AT-1 cell line and the highly metastatic Mat-LyLu line. However, the lowly metastatic AT-1 line had less motility as compared with the Mat-Lu and Mat-LyLu lines. This study revealed that metastatic potential could be partially predicted via area and shape factor and accurately predicted via cell motility.

Adhesion and invasion potential of rat prostatic cancer cells: correlation with metastatic potential.

This investigation examined the ability of low-metastatic AT-1 and high-metastatic ML and MLL cell lines to adhere to a monolayer of bovine aortic endothelial cells and fibronectin substratum and penetrate a matrigel basement membrane matrix. Cell area, shape factor, and motility as influenced by fibronectin were also examined using these cell lines. The ability of these cells to adhere to fibronectin and traverse matrigel matrix correlated with their metastatic potential. In addition, fibronectin increases cell circularity and reduces cell area as metastatic potential increases within AT-1 to ML or MLL cell lineages.

Expression and localization of androgen receptor in the R-3327 Dunning rat prostatic adenocarcinoma.

The Dunning R-3327 rat prostatic adenocarcinoma and its sublines have been developed as a model system to study prostate tumor progression. We have used this system to study the changes in androgen receptor (AR) and AR mRNA expression which occur during tumor progression from androgen dependent to androgen independent growth. Dorsal prostate and all tumor sublines contained a 10-kilobase AR mRNA on Northern blot analysis. The levels of AR mRNA in each subline compared to dorsal prostate (100%) were: H (75%) greater than G (48%) greater than HI (25%) greater than HI-F = AT-1 = AT-3 = MAT-Lu = MAT-Ly-Lu = less than 5%. Immunocytochemistry showed AR predominantly in acinar epithelial cells of dorsal prostate and the androgen sensitive H subline. In the H subline, both acinar epithelial cells and locally invasive adenocarcinoma cells within the stroma showed positive immunostaining. The androgen responsive, anaplastic G subline also showed strong positive immunostaining. The androgen resistant AT-1 and MAT-Lu sublines lacked immunostaining for the AR. Steroid autoradiography revealed a similar cellular distribution of AR. These data suggest that in the Dunning system the loss of androgen binding and responsiveness is primarily due to selective changes in gene expression and not to gene rearrangements or posttranscriptional or translational modification of the AR mRNA or protein.

Oncogene expression in prostate cancer: Dunning R3327 rat dorsal prostatic adenocarcinoma system.

Steady-state levels of myc, fos, p53, sis, and neu mRNAs were measured in eight variants derived from the Dunning R3327 rat prostate adenocarcinoma and compared to levels in normal dorsal prostate. Expression of the myb and erbB oncogenes in the Dunning tumors was below the limits of detection. Myc, p53, and sis mRNA levels in all tumors were at or above control levels. Fos mRNA levels were below control levels in four of five anaplastic tumors and were above control levels in the remaining tumors. A comparison of mRNA levels along the two Dunning lineages revealed that increased expression of these oncogenes did not correlate with tumor progression.

Expression of ras proto-oncogenes in the Dunning R3327 rat prostatic adenocarcinoma system.

Steady-state levels of c-Ha-ras mRNA were measured in eight sublines of the Dunning R3327 rat prostatic adenocarcinoma. As a control, normal dorsal prostate tissue was studied. Increased expression of c-Ha-ras is associated with tumor progression in one lineage of the Dunning R3327 system (H to AT1 to MAT-Lu and MAT-Ly-Lu). Here ras mRNA increases as the tumor advances from androgen dependence and a high degree of differentiation to an anaplastic aneuploid phenotype with high metastatic potential. However, in the other Dunning lineage (H to HI to HI-F to AT3), expression of c-Ha-ras is variable and does not correlate with tumor progression. Immunocytochemistry showed that levels of the c-Ha-ras p21 protein paralleled steady-state mRNA levels in variants. Transfection assays, using NIH/3T3 cells, suggested that the ras loci were not activated in the R3327 tumors. Levels of c-Ki-ras mRNA were also measured in the Dunning tumors; these did not correlate with tumor progression in either lineage. Expression of N-ras mRNA was not detected in the Dunning tumors.

The isolation, enrichment, and comparative electron microscopic characterization of cellular components of the aged rat ventral prostate.

Prostate cells were isolated from 3- and 23-month-old Sprague-Dawley rats. After sequential digestion with 0.1% collagenase at 37 degrees C, a mixed population of cells was obtained. The cells were layered on a five-step discontinuous Percoll gradient (g/ml: 1.024, 1.043, 1.048, 1.060, 1.089), centrifuged at 3,000 rpm X 30 minutes, which produced six distinct cellular subpopulations and a fibromuscular stroma (FMS). Electron microscopic characterization of the 3- and 23-month-old cellular subpopulations identified the following components, g/ml: debris and nonviable cells (1.020-1.025) nonsecretory epithelial cells (1.038-1.039), secretory epithelial cells (1.047-1.048), basal epithelial cells (1.057-1.059), differentiating epithelial cells (1.070-1.075), erythrocytes (1.085-1.089). This study demonstrates that one of the effects of age on the rat ventral prostate is an intracellular disorganization of isolated and enriched cellular components.

Gonococcal endocarditis in the antibiotic era.

Since the introduction of penicillin in 1942, there have been only 11 culture-proven cases of gonococcal endocarditis in the English literature. Most patients are under 30 years of age and have no history of heart disease. The aortic valve is often involved and aortic regurgitation is common. The bacteriologic diagnosis can be difficult and may require more than six blood cultures and a long incubation period. Circulating immune complexes appear to be the cause of many of the extracardiac manifestations. The three new cases reported herein and review of the literature emphasize the distinctive features of gonococcal endocarditis.