Java Web Start based software for automated quantitative nuclear analysis of prostate cancer and benign prostate hyperplasia

BACKGROUND: Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. METHODS: A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. RESULTS: The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. CONCLUSION: The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression

Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site

The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K(+) concentrations, with a sevenfold difference evident at 130 to 150 mM K(+). These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N

Androgen Receptor Expression and Cellular Proliferation During Transition from Androgen-Dependent to Recurrent Growth after Castration in the CWR22 Prostate Cancer Xenograft

Androgen receptor expression was analyzed in the CWR22 human prostate cancer xenograft model to better understand its role in prostate cancer recurrence after castration. In androgen-dependent tumors, 98.5% of tumor cell nuclei expressed androgen receptor with a mean optical density of 0.26 ± 0.01. On day 2 after castration androgen deprivation decreased immunostained cells to 2% that stained weakly (mean optical density, 0.16 ± 0.08). Cellular proliferation measured using Ki-67 revealed <1% immunostained cells on day 6. Androgen receptor immunostained cells increased to 63% on day 6 and 84% on day 32 although immunostaining remained weak. Cellular proliferation was undetectable beyond day 6 after castration until multiple foci of 5 to 20 proliferating cells became apparent on day 120. These foci expressed increased levels of prostate-specific antigen, an androgen receptor-regulated gene product. In tumors recurrent 150 days after castration androgen receptor-immunostaining intensity was similar to CWR22 tumors from intact mice although the percentage of cells immunostained was more variable. The appearance of proliferating tumor cells that expressed androgen receptor and prostate-specific antigen 120 days after castration suggests that these cells represent the origin of recurrent tumors