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Acid phosphatase activity biochemically in the primary tumor of 20 patients with prostatic carcinoma, was studied in an attempt to understand the basis for a correlation or lack of correlation between serum and/or bone marrow acid phosphatase levels and the presence and/or clinical behavior of prostatic carcinoma. The enzyme activity was similarly measured in 19 patients with benign prostatic hyperplasia as controls. On the average, enzyme activities were lower (P less than 0.002) in the tissues from patients with carcinoma. There was no correlation of enzyme activity in tumor with the age of the patient, stage of disease, degree of differentiation of the tumor, or serum acid phosphatase activity.

Aberrant crypt foci (ACF), the earliest neoplastic lesions of the colon, have genetic and epigenetic alterations. Loss of heterozygosity (LOH) of tumor suppressor gene loci is seen in most colon cancers, but it is not known how early in tumorigenesis this takes place. Nine microsatellite markers close to specific genes, that is, APC (5q21), PTPRJ (11p11), p53 (17p13) and DCC (18q21), were analyzed in 32 ACF and samples of normal crypts from the same 28 patients. Six losses of heterozygosity were found in 5 of 32 ACF: 4 losses of heterozygosity were at 11p11, the location of the gene for protein tyrosine phosphatase receptor type J (PTPRJ) and of a second independent region of deletion; the others were at 5q21 and 18q21. Microsatellite instability (MSI) with markers for a single locus was found in 4 of 32 ACF. All the observed allelic alterations (LOH and MSI) were in 8 of 32 ACF. The finding of LOH in ACF with normal expressions of adenomatous polyposis coli (APC) and beta-catenin proteins suggests that LOH can occur very early in colon neoplasia and perhaps even before APC mutations. The finding of 3 of 4 of the losses of heterozygosity at 11p11 for PTPRJ and half of all the losses of heterozygosity in this study at PTPRJ suggest that this gene plays a role early in colon neoplasia.

We have recently identified signal transducer and activator of transcription 5 (Stat5) as a critical survival factor for prostate cancer cells. We now report that activation of Stat5 is associated with high histological grade of human prostate cancer. Specifically, immunohistochemical analysis demonstrated a strong positive correlation with activation of Stat5 and high Gleason score in 114 human prostate cancers. To investigate the mechanisms underlying constitutive activation of Stat5 in prostate cancer, a dominant-negative mutant of Janus kinase 2 (Jak2) was delivered by adenovirus to CWR22Rv cells. Dominant-negative-Jak2 effectively blocked the activation of Stat5 whereas wild-type Jak2 enhanced activation, indicating that Jak2 is the main kinase that phosphorylates Stat5 in human prostate cancer cells. A ligand-induced mechanism for activation of Stat5 in prostate cancer was suggested by the ability of prolactin (Prl) to stimulate activation of both Jak2 and Stat5 in CWR22Rv human prostate cancer cells and in CWR22Rv xenograft tumors. In addition, Prl restored constitutive activation of Stat5 in five of six human prostate cancer specimens in ex vivo long-term organ cultures. Finally, Prl protein was locally expressed in the epithelium of 54% of 80 human prostate cancer specimens with positive correlation with high Gleason scores and activation of Stat5. In conclusion, our data indicate that increased activation of Stat5 was associated with more biologically aggressive behavior of prostate cancer. The results further suggest that Jak2 is the principal Stat5 tyrosine kinase in human prostate cancer, possibly activated by autocrine/paracrine Prl.

Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.

Background: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. Methods: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. Results: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft ; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently over-expressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P = .0001). Conclusions: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.

BACKGROUND Established cell lines or xenografts from prostatic carcinoma have been infrequently studied cytogenetically. CWR22 and CWR22-R are xenografts that are unique in offering one strongly androgen-dependent and several relapsed strains of a human prostate cancer that can be investigated in the laboratory. We report on the cytogenetic characterization of the hormone-dependent CWR22, and the relapsed CWR22-R serially transplanted xenografts, in our laboratory. METHODS We utilized a suspension harvest of the xenograft tissue to optimize our yield for metaphase chromosome studies and analyzed the hormone-dependent CWR22 and four relapsed CWR22-R xenografts. These studies were accomplished using standard G-banded analysis and fluorescence in situ hybridization (FISH). A variety of DNA probes including α-satellite DNA probes, and chromosomal libraries, were utilized for the FISH analysis. RESULTS Utilizing both standard cytogenetic analysis and FISH studies we have more precisely defined the CWR22 xenograft: 49,XY,+i(1)(q10),−2,der(4)t(2;4)(p21;q33), +7,+8,+12[7]/50,XY,idem,+der(2)t(2;4)(p21;q33)del(2)(q13q33)[13]. Four relapsed xenografts, CWR22R-2152, CWR22R-2524, CWR22R-2274, and CWR22R-2272 were also studied. Each of these lines demonstrated a different karyotype. CONCLUSIONS The CWR22 karyotype offers the simplest reported karyotype for a prostate cancer tissue culture cell line or xenograft; this makes CWR22 an attractive candidate for studies of genetic changes associated with the relapse of prostate cancer treated with androgen withdrawal. Four separate, serially transplanted, relapsed CWR22-R xenografts were detected, each with a separate karyotype. Prostate 41:7–11, 1999. © 1999 Wiley-Liss, Inc.

A cell line has been derived from a human prostatic carcinoma xenograft, CWR22R. This represents one of very few available cell lines representative of this disease. The cell line is derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft. Flow cytometric and cytogenetic analysis showed that this cell line represents one hyper DNA-diploid stem line with two clonal, evolved cytogenetic sublines. The basic karyotype is close to that of the grandparent xenograft, CWR22, and is relatively simple with 50 chromosomes. In nude mice, the line forms tumors with morphology similar to that of the xenografts, and like the parental CWR22 and CWR22R xenografts, this cell line expresses prostate specific antigen. Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis. Growth is stimulated by epidermal growth factor but is not inhibited by transforming growth factor-beta1.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) are important modulators of IGF action in many tissues including human prostate. IGFBPs and the androgen receptor (AR) are expressed in CWR22, an androgen-dependent epithelial cell human CaP xenograft that retains biological characteristics of human CaPs, including regression following androgen withdrawal and recurrent growth of AR-containing cells in the absence of testicular androgens beginning several months after castration. Northern blot and in situ hybridization analyses demonstrated that IGFBP-5 is androgen-regulated in CWR22. IGFBP-5 messenger RNA (mRNA) decreased by 90% following castration of tumor-bearing mice compared with noncastrate androgen-stimulated mice. Testosterone treatment of CWR22 tumor-bearing mice 6 or 12 days after castration increased IGFBP-5 mRNA 10- to 12-fold. Levels of other IGFBP mRNAs did not change following androgen withdrawal and replacement. IGFBP-5 protein in tumor extracts bound 125I-labeled IGF-I in ligand blot assays and the amounts of IGFBP-5 measured by immunoblotting paralleled the levels of IGFBP-5 mRNA. Androgen-induced expression of IGFBP-5 was at a maximum level within 24 h after testosterone replacement, whereas the major increase in cell proliferation as measured by Ki-67 immunostaining occurred between 24-48 h. This time course suggested IGFBP-5 may be a mediator of androgen-induced growth of CWR22. In tumors that recurred several months following castration, IGFBP-5 mRNA and protein increased to levels that approached those in androgen-stimulated CWR22 tumors from noncastrate mice. IGFBP-5 immunohistochemical staining of prostate tissue specimens from patients was stronger in androgen-dependent and androgen-independent CaP than in areas of intraepithelial neoplasia (PIN) or benign prostatic hyperplasia (BPH). IGFBP-5 mRNA in these specimens was localized predominantly to stromal cells and IGFBP-5 protein to epithelial cell membranes.

Purpose: The expression of the cyclin dependent kinase inhibitors p21 and p27 was examined in prostatic adenocarcinomas following castration.Materials and Methods: Male nude mice inoculated with the androgen dependent human prostatic tumor CWR22 were castrated when the tumors reached a volume of 0.8 to 1.1 cm.3 and were sacrificed at 3, 7, 21, 28 and 42 days post-castration. An additional group of mice received a subcutaneous testosterone pellet at 21 days post-castration and was sacrificed at 28 days post-castration. The expression of the Ki-67 antigen, p21 and p27 was examined by immunohistochemistry.Results: The mitotic rate as well as the number of Ki-67 antigen positive cells decreased to 3% of intact control values by 7 days post-castration and were less than 0.01% of intact control values at 21, 28 and 42 days post-castration. The percentage of p21 expressing cells decreased from 15 +/− 2% in intact controls to less than 1% by 42 days post-castration. In contrast, the percentage of cells th...

BACKGROUND Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation. METHODS Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts. RESULTS Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 trasnscripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined. CONCLUSIONS These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology. Prostate 38:40–45, 1999. © 1999 Wiley-Liss, Inc.

Microscopic evaluation of whole-mount colons stained with methylene blue and/or hexosaminidase has identified putative preneoplastic lesions in the colons of rodents treated with carcinogen and in the grossly normal colons of humans. Enzyme histochemistry with glycol methacrylate sections has permitted the identification of putative premalignant lesions in rodent livers, human and rodent colons, and human prostates. Immunohistochemistry with paraffin-embedded tissues has been used to identify and characterize putative premalignant lesions in human colons and prostates.

Background In men with prostate cancer, the gland usually contains two or more widely separate tumors. A critical issue of prostatic carcinogenesis is whether these multiple tumors are independent in origin. Molecular analysis of microsatellite (i.e., highly repeated, short nucleotide sequences) alterations in the DNA from separate tumors in the same prostate can be used to determine whether or not these separate tumors arise independently. Methods Four microsatellite polymorphic markers (D8S133, D8S136, and D8S137, for a putative tumor suppressor gene on chromosome 8p, and D17S855, for the BRCA1 gene on chromosome 17q) were used to examine the pattern of allelic loss in prostate cancer from 19 patients who had two or more distantly separate tumors (i.e., located on contralateral sides or separated by at least half the anterior-posterior diameter of the prostate). Forty distantly separate tumors were microdissected, DNA samples were prepared from formalin-fixed, paraffin-embedded wholemount prostate tissue section, and the overall frequencies of loss of heterozygosity at the four loci were determined. Results The pattern of allelic loss was compatible with independent tumor origin in 15 of 18 informative cases. A random discordant pattern of allelic deletion was observed in distantly separate tumors, whereas the same allele was consistently lost in cells from different regions of the same tumor. For three patients, the results were compatible with either intraglandular dissemination or independent origin of prostate cancer. Conclusions Our data suggest that multiple tumors in some patients with prostate cancer have independent origin.

Prostate carcinoma (PCA) is the most commonly diagnosed malignancy in American men. Our knowledge of PCA growth regulation lags behind that of other cancers, such as breast and colon carcinomas. Among receptor tyrosine kinases, the ErbB family is most frequently implicated in neoplasia. We report here the expression of ErbB family kinases and their ligands in PCA cell lines and a xenograft. While ErbB1/EGFR, ErbB2/NEU, and ErbB3 were always observed in a distinct pattern, ErbB4 was not observed. Interestingly, while TGF-alpha was expressed in the majority of PCA lines, the ligand Neu Differentiation Factor/Heregulin (NDF) was expressed only in an immortalized, non-transformed prostate epithelial line. Concomitantly, there was a significant difference in biological response to these ligands. NDF inhibited LNCaP growth and induced an epithelial-like morphological change, in contrast to TGF-alpha, which accelerated cell growth. We also performed the first comprehensive analysis of NDF signaling in a prostate line. LNCaP stimulated with NDF demonstrated crosstalk between ErbB3 and ErbB2 which did not involve ErbB1. NDF also turned on several cascades, including those of PI3-K, ERK/MAPK, mHOG/p38 and JNK/SAPK, but not those of PLCgamma or the STAT family. This signaling pattern is distinct from that of TGF-alpha. The activation of mHOG by ErbB2 or ErbB3 has not been reported, and may contribute to the unusual phenotype. PI3-K activation is characterized by the formation of a striking 'activation complex' with multiple tyrosine-phosphorylated species, including ErbB3. Our studies provide a framework in which to dissect the growth and differentiation signals of prostate cancer cells.

An androgen receptor (AR) gene mutation identified in the androgen-dependent human prostate cancer xenograft, CWR22, changed codon 874 in the ligand-binding domain (exon H) from CAT for histidine to TAT for tyrosine and abolished a restriction site for the endonuclease SfaNI. SfaNI digestion of AR exon H DNA from normal but not from prostate cancer tissue indicated H874Y is a somatic mutation that occurred before the initial tumor transplant. CWR22, an epithelial cell tumor, expresses a 9.6-kb AR mRNA similar in size to the AR mRNA in human benign prostatic hyperplasia. AR protein is present in cell nuclei by immunostaining as in other androgen-responsive tissues. Transcriptional activity of recombinant H874Y transiently expressed in CV1 cells in the presence of testosterone or dihydrotestosterone was similar to that of wild type AR. With dihydrotestosterone at a near physiological concentration (0.01 nM), H874Y and wild type AR induced 2-fold greater luciferase activity than did the LNCaP mutant AR T877A. The adrenal androgen, dehydroepiandrosterone (10 and 100 nM) with H874Y stimulated a 3- to 8-fold greater response than with wild type AR and at 100 nM the response was similar with the LNCaP mutant. H874Y, like the LNCaP cell mutant, was more responsive to estradiol and progesterone than was wild type AR. The antiandrogen hydroxyflutamide (10 nM) had greater agonist activity (4- to 7-fold) with both mutant ARs than with wild type AR. AR mutations that alter ligand specificity may influence tumor progression subsequent to androgen withdrawal by making the AR more responsive to adrenal androgens or antiandrogens.

High-resolution profiles of the reflections of a broad-band 4 MHz ultrasound pulse from radical prostatectomy and autopsy specimens in vitro have been captured and analyzed. An attempt was made to correlate the histopathology of each prostate reflection site with the profile of the pulse reflected from that site. Reflections from 125 sites on 22 prostate specimens were studied. Although the reflected pulse from each site displayed a distinctive profile, we could find no obvious correlation between the shape of that profile and the histologic findings at that site.

Aberrant crypt foci are putative preneoplastic lesions found in the colons of carcinogen-treated rodents and at an increased frequency in humans at increased risk for colon cancer. There is a strong association between aberrant crypt foci and colon cancer, including many shared phenotypic and genetic alterations. The aim of this study is to present further evidence of a relationship between aberrant crypt foci and colon cancer in humans. Multiple aberrant crypt foci from a single patient were identified in unembedded colonic mucosa. Histological sections of the aberrant crypt foci and adjacent mucosa were evaluated for dysplasia, proliferative activity, and pigment-laden macrophages that were characterized with histochemical techniques. The first patient with sporadic colon cancer identified with aberrant crypt foci with carcinoma in situ is described. It is interesting that this 99-year-old patient had multiple carcinomas in situ, pseudomelanosis coli, and two metachronous colon cancers. These data lend support to the hypothesis that aberrant crypt foci are precursors of some colon cancers.

This study evaluated the growth of human prostate tumors in histoculture, an in vitro culture technique that maintains three-dimensional tissue structure and organization. Eighty-six percent of 50 tumor specimens from 50 patients were successfully cultured. The histocultures showed proliferation of epithelial tumor cells and stromal cells. Prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) were detectable by immunohistochemistry for at least 8 weeks. Synthesis of PSA was further confirmed by its presence in medium. The mean thymidine labeling index (LI) of the neoplastic cells was 62 percent. There was no correlation between thymidine LI and tumor grade. The explants maintained their characteristics for at least 8 weeks as indicated by unchanged thymidine LI, PSA and PAP immunohistochemistry after 2 and 8 weeks in culture. A 24 to 48 hour delay in processing the tissues for culture did not reduce the thymidine LI.The thymidine LI and PSA and PAP staining in tumors cultured in a M...

Background. During the past decade, the National Cancer Institute became aware of a lack of availability of human tissues for research, especially in the fields of molecular biology, genetics, and immunology. Methods. In 1987, the National Cancer Institute (NCI) established the Cooperative Human Tissue Network (CHTN) by funding three institutions that had extensive experience in the procurement and distribution of tissues for research. Results. Since its inception, the CHTN has been expanded to five member institutions, with the addition of the Columbus Children's Hospital (the Pediatric Division for the procurement of pediatric tissues) and Case Western Reserve University. Each of the five divisions have established regional networks of institutions that supply tissues to the major divisions. From 1987 through 1991, the CHTN has distributed more than 29,000 human tissue samples and supplied approximately 500 researchers throughout the country. Conclusions. This report describes the development and current status of the CHTN, a valuable resource to the research community.

Aberrant crypts are recognized in methylene blue-stained, unsectioned, colonic mucosa by their increased size, elliptical lumenal opening, thicker epithelial layer, and increased pericryptal region. Aberrant crypt foci in rodents are observed as early as 2 weeks and for at least 9 months after a single dose of carcinogen, have a distribution that parallels that of tumors, and have an increased number of aberrant crypts per focus with time after the carcinogen dose. The ability to quantify these lesions in the entire colon of rodents in less than an hour suggests that aberrant crypts may provide a highly efficient in vivo bioassay for colon carcinogens. Since aberrant crypt foci appear to be the earliest identifiable putative precursors of colon cancer, they represent lesions that can be characterized further for the earliest genetic and biochemical alterations. In F344 rats, we have demonstrated that aberrant crypts have multiple histochemically-detectable enzyme alterations. Using similar techniques, we were the first to demonstrate aberrant crypts in unsectioned human mucosa. After embedding and sectioning, these microscopic aberrant crypts resemble rare lesions described earlier in the literature after extensive serial sectioning. In rats and humans, aberrant crypts may be histologically normal or display varying degrees of dysplasia and histochemically-detectable altered enzyme activities. These putative, preneoplastic lesions should reveal early changes that precede colon cancer and ways to alter their progression.

Aberrant crypt foci (ACF), the earliest identified monoclonal lesions in the colon, provide insights into changes that promote and/or accompany the transformation of normal colonic epithelial cells to colorectal cancer. Fatty acid synthase (FAS), the primary enzyme involved in de novo lipogenesis from carbohydrates, is expressed at low levels in most normal human tissues but is elevated in several human neoplasms including colorectal adenomas and carcinomas. To mdetermine if this pathway is altered even earlier in colorectal tumorigenesis, 35 human ACF from 21 patients were evaluated for the immunohistochemical expression of FAS. Sections of colon cancer served as positive controls, and normal colonic mucosa distant from cancer or ACF served as negative controls. FAS expression was increased in 30 (86%) ACF compared with that in adjacent normal colonic mucosa. The expression of FAS in ACF was not related to the degree of dysplasia or to the number of crypts in the ACF. The over expression of FAS in a high proportion of ACF suggests that this enzyme plays an important role very early in colorectal tumorigenesis and may be a target for chemoprevention.

Extracts of tissue showed that prostatic carcinomas contain less leucine aminopeptidase activity than benign prostatic hyperplasia. This is true when activity is expressed as specific activity (P = 0.0033), specific activity/% epithelium (P = 0.0007), activity/wet weight of tissue (P = 0.0028), or activity/wet weight of tissue/% epithelium (P = 0.0005). Almost all histochemically demonstrable activity is located in the epithelium. Enzymatic activities in extracts and in histochemical preparations showed similar differences between carcinoma and benign prostatic hyperplasia and were related (B = -0.38, P = 0.0400) to Gleason's grades. The transurethrally resected prostate cancers studied contained no well-differentiated tumors and a high proportion of poorly differentiated tumors. Histochemical activity is absent in most prostatic carcinomas and decreased in others. This observation is particularly interesting in view of the growing knowledge of tumor suppressor genes.

Among the many approaches to the separation of cells, sedimentation either at unit gravity or with a centrifugal force has been widely used. Since the quality of the suspensions of cells prior to sedimentation greatly influences the separation, it is important to evaluate several methods for the dispersion of solid tissues to obtain suspensions of viable, single cells. The optimal approach to a particular tissue varies as a function of organ, donor species and age, and other experimental variables. There are two kinds of sedimentation that can be used for cell separation in continuous gradients: velocity sedimentation and isopycnic sedimentation. Isopycnic sedimentation accomplishes cell separations according to the different densities of cells. There are four commonly used forms of velocity sedimentation: sedimentation at unit gravity, isokinetic sedimentation, sedimentation in a reorienting zonal rotor, and elutriation. The choice of method for the separation of particular kinds of cells will depend on the needs of the investigator, including the number of cells desired, the cost, and the need for work under sterile conditions.

The analysis of antigens, enzyme histochemical markers, and DNA has become an important part of the classification of some leukemias, lymphomas, and other neoplastic diseases. Many of the relevant antigens and most of the relevant enzyme histochemical activities are destroyed and others are less than optimally preserved in tissues embedded in hot paraffin. Most enzymatic activities and antigens are well preserved in tissues embedded at 4 degrees C in glycol methacrylate (GMA). The measurement of DNA content in neoplastic cells with the most commonly employed techniques depended on the availability of fresh suspensions of cells until the development by Hedley of methods that permit the recovery of nuclei from paraffin blocks for this purpose. In order to facilitate the analysis of antigens, enzymatic markers, and DNA from the same sample of tissue, we have developed a means of recovery of nuclei from GMA-embedded tissues. Twenty-microns-thick sections of GMA- embedded tonsil were either pretreated with an organic solvent (absolute ethanol or 2-ethoxyethanol) followed by rehydration in phosphate buffered saline (PBS) or directly rehydrated in PBS. The suspensions were formed mechanically by gentle sonication. The type of fixative and length of PBS rehydration were varied. Tissue fixed in 100% acetone, embedded in GMA, and rehydrated directly in PBS for six days gave the highest average yield of nuclei, 3.7 x 10(7) nuclei per gram tissue. In order to assess DNA binding of fluorescent dyes, 2- microns-thick GMA sections were stained with chromomycin, Hoechst 33342 (Sigma Chemical, St Louis, MO), and propidium iodide. Hoechst 33342 bound specifically to the nuclei with low background staining.

For the study of micrometastases at their earliest stages, we transfected the lacZ gene, which codes for beta-D-galactosidase in Escherichia coli, into BALB/c 3T3 cells transformed by the Ha-ras oncogene (also known as HRAS1) of a human EJ bladder carcinoma. These cells were subsequently injected into 6-week-old, female athymic NCR-NU nude mice by several routes. With chromogenic detection of the product of the lacZ gene (a heterologous gene not observed in animal cells) by use of 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside, we easily identified tumor cells implanted in the lungs minutes after intravenous injection by the intensely blue staining of the cells harboring the lacZ gene. The number of lung-associated tumor cells remained constant for several hours after intravenous injection but then decreased to a stable level by 24 hours. At most sites of lung invasion, multiple tumor cells, rather than single cells, were identified; this finding suggests that cooperation among multiple cells may be important in the early stages of micrometastasis development. Within several days, a few foci of micrometastases were expanding by proliferation and/or migration of individual tumor cells among host lung cells. These results confirm that the lacZ gene is an ultrasensitive histochemical marker for analyzing both qualitatively and quantitatively the earliest stages of micrometastasis development in the lung and in other organs where micrometastases may ensue.

Previously, we reported that the activities of several enzymes extracted from prostatic cancers were closely related to the histologic grades (Gleason's grades) of the cancers. The survival of patients was more closely correlated with certain enzymatic activities than with Gleason's grades. Unfortunately, because our assays required relatively large amounts of tissue, the increasing proportion of patients who have needle biopsies as their only surgical procedure had to be excluded from our previous study. We now report a method that permits multiple enzyme assays to be carried out with much smaller amounts of tissue. This method will enable us to avoid the selection inherent in the exclusion of patients who have only needle biopsies and should permit the prospective study of most patients with prostatic carcinoma.

We are developing in vivo small animal imaging techniques that can measure early effects of photodynamic therapy (PDT) for prostate cancer. PDT is an emerging therapeutic modality that continues to show promise in the treatment of cancer. At our institution, a new second-generation photosensitizing drug, the silicon phthalocyanine Pc 4, has been developed and evaluated at the Case Comprehensive Cancer Center. In this study, we are developing magnetic resonance imaging (MRI) techniques that provide therapy monitoring and early assessment of tumor response to PDT. We generated human prostate cancer xenografts in athymic nude mice. For the imaging experiments, we used a high-field 9.4-T small animal MR scanner (Bruker Biospec). High-resolution MR images were acquired from the treated and control tumors pre- and post-PDT and 24 hr after PDT. We utilized multi-slice multi-echo (MSME) MR sequences. During imaging acquisitions, the animals were anesthetized with a continuous supply of 2% isoflurane in oxygen and were continuously monitored for respiration and temperature. After imaging experiments, we manually segmented the tumors on each image slice for quantitative image analyses. We computed three-dimensional T2 maps for the tumor regions from the MSME images. We plotted the histograms of the T2 maps for each tumor pre- and post-PDT and 24 hr after PDT. After the imaging and PDT experiments, we dissected the tumor tissues and used the histologic slides to validate the MR images. In this study, six mice with human prostate cancer tumors were imaged and treated at the Case Center for Imaging Research. The T2 values of treated tumors increased by 24 ± 14% 24 hr after the therapy. The control tumors did not demonstrate significant changes of the T2 values. Inflammation and necrosis were observed within the treated tumors 24 hour after the treatment. Preliminary results show that Pc 4-PDT is effective for the treatment of human prostate cancer in mice. The small animal MR imaging provides a useful tool to evaluate early tumor response to photodynamic therapy in mice.

Mark E. Stearns (Chairperson),1* Joy L. Ware (Rapporteur),2 David B. Agus,3 Ching-Jer Chang,4 Isaiah J. Fidler,5 Rose S. Fife,6 Robin Goode,7 Eric Holmes,8 Michael S. Kinch,9 Donna M. Peehl,10 Thomas G. Pretlow II,11 and George N. Thalmann12 1Department of Pathology, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 2Department of Pathology, Virginia Commonwealth University, Richmond, Virginia 3Memorial Sloan-Kettering Cancer Center, New York, New York 4Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 5Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 6Indiana University School of Medicine, Indianapolis, Indiana 7Lilly Research Laboratories, Indianapolis, Indiana 8Pacific Northwest Cancer Foundation, Seattle, Washington 9Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 10Department of Urology, Stanford University, Stanford, California 11Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 12Universitat Bern, Bern, Switzerland

Since aberrant crypt foci (ACF) were first described in 1987, they have been the subjects of hundreds of papers; however, the debate continues about their role in colorectal tumorigenesis. This review focuses on the many phenotypic, genetic and epigenetic alterations in ACF that support the hypothesis that ACF are putative precursors of colorectal cancer in both humans and experimental animals. Human ACF, both with and without dysplasia, are monoclonal and display evidence of chromosomal instability. Both of these characteristics are shared by colorectal cancers. While most ACF do not have APC mutations, a large proportion has KRAS mutations and methylated SFRP1 and SFRP2 genes. This epigenetic inactivation gives rise to constitutive Wnt signaling in these putative precursors of colorectal cancer.

Most research that requires long-term propagation of prostate cancer (PCA) cells is carried out with three cell lines: DU 145, PC-3, and LNCaP. All but one of these lines, LNCaP, fail to express prostate-specific antigen (PSA), androgen receptors, and/or any other prostate phenotype. Some prostatic fluids contain prostate cancer cells. The goals of this research were as follows: (1) to test the tumorigenicity of, and to develop transplantable xenografts from, PCA cells in prostatic fluid; (2) to develop methods for enhancing the tumorigenicity of small numbers of these PCA cells without deliberately altering their genes; (3) to test these methods for enhancement of tumorigenicity with prostatic fluid cells; and (4) to initiate clinical followup. As detailed in the body of the report, the aims of the research have been limited by exceptionally weak clinical collaboration that has become progressively weaker. The fluids that the author tested earlier were promising, but samples tested in the past year have been quite inadequate. Co-injection of lethally irradiated, growth-factor-producing cells was encouraging in some experiments with some tumors, but results were quite variable in repeated experiments. The inadequacies in fluid samples and the repeatability of experiments are detailed in this report. The author concludes that, prostate cancer cells from prostatic fluids of a significant proportion of patients with primary prostate cancers can survive for a few months in nude mice, and they do not grow rapidly enough to form transplantable xenografts with the technology that is currently available.

CWR22 has been a valuable xenograft model for the study of prostate cancer progression from an androgen-dependent tumor to one that grows in castrated animals. Herein, we report the identification and characterization of a novel androgen receptor (AR) mutation occurring in a relapsed tumor (CWR22R-2152) and in the CWR22Rv1 cell line established from it. The mutation was not detected in the original, hormone-dependent CWR22 xenograft, indicating that this change occurred during the progression to androgen independence. It is characterized by an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA-binding domain. Accordingly, immunoblot analyses demonstrated the expression of an AR species having an approximately 5-kDa increase in size relative to the LNCaP AR. This was accompanied by a COOH-terminally truncated AR species migrating with a relative mass of 75-80 kDa, referred to as ARDeltaLBD because it lacks the ligand-binding domain. By recreating the exon 3 duplication mutation in a wild-type AR expression construct, the generation of ARDeltaLBD could be recapitulated. Whereas ARDeltaLBD exhibited constitutive nuclear localization and DNA binding, these functions in the full-length AR remained androgen dependent. The CWR22Rv1 AR repertoire displayed dose-dependent, androgen-responsive transcriptional transactivation in reporter assays, albeit to a lesser extent in comparison with LNCaP. This cell line also expressed low levels of prostate-specific antigen mRNA and did not express or secrete detectable levels of prostate-specific antigen protein in androgen-depleted medium or in response to physiological androgenic stimulation. In summary, the CWR22Rv1 cell line displays both androgen-responsive and androgen-insensitive features due, at least in part, to a novel insertional mutation of the AR.

The tissue kallikreins (KLKs) form a family of serine proteases that are involved in processing of polypeptide precursors and have important roles in a variety of physiologic and pathological processes. Common features of all tissue kallikrein genes identified to date in various species include a similar genomic organization of five exons, a conserved triad of amino acids for serine protease catalytic activity, and a signal peptide sequence encoded in the first exon. Here, we show that KLK4/KLK-L1/prostase/ARM1 (hereafter called KLK4) is the first significantly divergent member of the kallikrein family. The exon predicted to code for a signal peptide is absent in KLK4, which is likely to affect the function of the encoded protein. Green fluorescent protein (GFP)-tagged KLK4 has a distinct perinuclear localization, suggesting that its primary function is inside the cell, in contrast to the other tissue kallikreins characterized so far that have major extracellular functions. There are at least two differentially spliced, truncated variants of KLK4 that are either exclusively or predominantly localized to the nucleus when labeled with GFP. Furthermore, KLK4 expression is regulated by multiple hormones in prostate cancer cells and is deregulated in the androgen-independent phase of prostate cancer. These findings demonstrate that KLK4 is a unique member of the kallikrein family that may have a role in the progression of prostate cancer.

Nuclear receptors form a superfamily of ligand-activated transcription factors. In contrast to the significant advances made in recent years to dissect nuclear receptor structure and their corresponding function, progress has been rather slow in the identification of target genes for nuclear receptors, information that is a prerequisite for understanding hormone action. Here, we describe a simple screening protocol that makes it possible to efficiently and effectively clone hormone-responsive genes that are specific to a tissue of interest. When this procedure was used to clone prostate-specific and androgen-responsive genes, approximately 40% of the clones selected at random represented genes that are known to be androgen regulated and are largely specific to prostate for expression, such as prostate specific antigen (PSA). A further 37% are known to be highly enriched in prostate for expression, but their androgen regulation is yet to be studied. The rest of the clones represented novel genes, expressed sequence tags, or known genes whose possible androgen regulation has not yet been assessed. This screening scheme can be applied to any hormone/ligand to clone differentially expressed genes specific to a tissue of interest. Identification of such genes and their characterization should greatly facilitate understanding hormone action in normal and pathological conditions.

Background The RET proto-oncogene encodes a protein that belongs to the tyrosine kinase growth factor receptor family. Germline point mutations in RET are found in individuals with multiple endocrine neoplasia (MEN) syndromes, and gene rearrangements have been reported in papillary thyroid cancers. We recently identified transcripts of the RET proto-oncogene in human prostate cancer xenografts and prostate cancer cell lines by means of reverse transcription-polymerase chain reaction analyses. The purpose of this study was to investigate Ret protein expression in human prostate tissue. Methods Ret protein expression was evaluated immunohistochemically in formalin-fixed, paraffin-embedded whole-prostate sections. The prostate specimens were obtained from 30 patients with prostate cancer after radical prostatectomies. Ret protein expression was compared in tumor foci and benign prostatic tissue. Medullary thyroid carcinoma tissue associated with an MEN syndrome and papillary thyroid cancer tissue served as positive controls. Results Ret appeared to be overexpressed in high-grade (histopathologically advanced) prostatic intraepithelial neoplasia (PIN) and prostate cancer when compared with its expression level in benign prostatic secretory epithelium. In addition, there was an apparent increase in Ret protein expression with decreased cellular differentiation, i.e., increasing Gleason pattern. Conclusion Expression of the RET proto-oncogene in benign prostatic epithelium, high-grade PIN, and histopathologically advanced prostate cancer suggests that RET may play a role in the growth of both benign and neoplastic prostate epithelial cells.

Lack of well-defined relevant in vivo or in vitro tumor models is one of the major limitations in assessing candidate therapeutic regimens, especially gene therapy, for prostate cancer. Since gene therapy is emerging as a potentially powerful therapeutic modality, it is desirable to evaluate this approach for the treatment of human prostate cancer.We sought to establish a relevant ex vivo tumor model for gene therapy studies of human prostate cancer.We constructed and established a transgenic human tumor model consisting of three major components: 1) human primary prostate cancer cells, CWR22, reactivated for growth after storage in liquid nitrogen; 2) a collagen gel ex vivo tissue culture system useful for short-term maintenance and manipulation of CWR22 cells under in vitro experimental conditions; and 3) a high-velocity, particle-mediated gene transfer system that is highly efficient in the ex vivo transfection of target cells. Prostate-specific antigen (PSA) levels in the cell culture media were monitored after transfecting CWR22 cells with candidate therapeutic genes, including the cytokines human interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), both as complementary DNAs [cDNAs]). CWR22 cells, transfected with firefly luciferase cDNA as a reporter gene, served as control cells for cytokine gene expression. CWR22 cells, transfected with the bacterial beta-galactosidase cDNA as a reporter gene, were used to assess the efficiency of gene transfer. Transcription of each of the cDNAs was driven by the cytomegalovirus (CMV) early gene promoter.The three-dimensional organization of tumor cells and functional characteristics of human prostate cancers were maintained in this ex vivo model of prostate cancer. Candidate therapeutic genes, CMV-IL-2 and CMV-GM-CSF, were expressed at peak levels of up to 38 ng of protein per 10(6) cells every 24 hours. IL-2 and GM-CSF secretion was sustained at approximately 40%-50% of peak levels during the entire experimental period (9-10 days in culture). At 7 days after gene delivery, a more than twofold reduction in the secretion of PSA was detected in the IL-2 (3.8 +/- 1.3 ng/10(4) cells every 24 hours [mean +/- standard deviation]) or GM-CSF (4.0 +/- 1.7 ng/10(4) cells every 24 hours) cDNA transfected cells as compared with the control cells transfected with luciferase cDNA (9.3 +/- 1.0 ng/10(4) cells every 24 hours). Up to 10% of the cells transfected with beta-galactosidase cDNA expressed measurable beta-galactosidase activity.This study demonstrated an efficient, rapid, and reliable system for gene transfer and expression in primary human prostatic carcinoma cells maintained in a collagen gel culture system.Our findings suggest a broad application of this CWR22 xenograft primary culture system as an ex vivo tumor model for the evaluation and characterization of various candidate therapeutic genes for human prostate cancer gene therapy, including a cytokine gene-modified tumor vaccine strategy.

Summary Preneoplastic lesions, early neoplastic lesions and carcinomas in situ have been demonstrated to be of great value for many purposes in many organ systems. Their recognition can be useful in epidemiological studies and can facilitate the selection of patients for therapeutic interventions. They can be used as “surrogate endpoint biomarkers” in studies aimed at the chemoprevention of cancers. In the lung, colon and various other organs, such markers are well recognized to be associated with the development o f cancer in man. In the livers and colons of experimental animals, there has been detailed characterization of “enzyme-altered foci” (EAF) as “putative preneoplastic markers.” The words “surrogate” and “putative” are important; the biological potential of these lesions needs to be elucidated in much greater detail. The quantification of early lesions that are associated with and sometimes precursors of neo plasia is of particular value because they are much more numerous, in most organ systems, than the carcinomas that develop in the same organs. The most abundant of these lesions show minimal or no morphological alterations. For example, EAF and aberrant crypts are more numerous than polyps in the colons of patients and experimental animals with cancer or precancerous conditions that affect the colon. Currently, there are few well documented putative or surrogate markers that are highly associated with the development of Prostatic carcinoma in man. The best documented among these is Prostatic intraepithelial neoplasia. We have recently reported the identification of EAF in the human prostate. While they share many phenotypic alterations with Prostatic intraepithelial neoplasia, much remains to be accomplished if their biological fate is to be understood.

Organ culture of the human prostate began in the 1970s and was modeled after the work of Lasnitzki and her collaborators in the mouse two decades earlier. In organ culture of human prostates, one sees a rapid increase in epithelial cells and decrease in stromal cells during the first 3–5 days of culture. While modulation of many phenotypic properties occurs, these cultures provide a simple and rapid way to achieve large numbers of human prostatic epithelial cells in cultured tissues that are markedly depleted of stromal cells. There is some evidence that organ cultures are maintained in slightly better functional states in the presence of androgens; however, most of this evidence is less than quantitative. Most organ culture of prostates has been accomplished with tissues from unspecified locations within the prostate; interpretation of cultures carried out in this fashion has been less complete than would have been possible if they had been carried out from specific anatomic locations within the prostate. Careful pathological characterization of locations contiguous to the cultured tissue is mandatory if cultures are to be interpreted meaningfully. © 1995 Wiley-Liss, Inc.

Background Prostatic carcinoma is both the most common invasive cancer and the second most common cause of cancer deaths in men in the United States. Before 1991, attempts to propagate prostatic carcinoma from primary tumors for periods longer than 3 months were unsuccessful in vivo and in vitro with rare exceptions. In 1991, we reported establishment of slowly growing tumors for six of 10 human primary prostatic carcinomas approximately 2-6 months after transplantation. However, none of the tumors were larger than 5 mm or serially transplantable. Purpose Our purpose in this study was to determine whether human primary prostatic carcinoma could be grown as serially transplantable xenografts. Methods Cells from primary prostatic carcinomas obtained from transurethral prostatic resections or total prostatectomies in 20 patients were injected subcutaneously into male nude mice on the day of surgery. Sustained-release testosterone pellets were placed subcutaneously in the mice 2-24 days before transplantation of tumors and at intervals of 10-12 weeks. Serial transplantations in subsequent generations of mice were carried out by similar methods. Chromosome analysis was performed on six tumors. Results Six of 20 primary prostatic carcinomas have grown sufficiently to permit serial transplantation into second mice; four have been documented histopathologically in the second mouse and serially transplanted into three or more successive mice. When a single primary tumor was injected into several mice by the same procedure, tumors failed to grow in some recipients but became serially transplantable in others. Growth of these tumors is slow and irregular, with frequent regressions. Short-term cultures of 10 tumors, eight of which were injected into mice in parallel, were initiated on the day of surgery; CWR31, which was successfully transplanted serially, exhibited only aberrant metaphases and showed clonal, chromosomal changes in culture. Including CWR31, three of the six tumors for which chromosomal analysis was successful contained clonal aberrations. Preliminary studies of SCID (severe combined immunodeficient) mice suggest that they are not superior to nude mice for establishment of serially transplantable prostatic carcinoma xenografts. Conclusions A proportion of human primary prostatic carcinomas can be grown as xenografts. Four new serially transplantable xenografts (CWR21, CWR31, CWR91, and CWR22) are currently propagated in our laboratory, a resource that was not previously available. Implications Our experience suggests that the most important factor in serial transplantation is the collaboration of urologists and pathologists in expediting placement of the tumor in cold saline, examination of the frozen section, and transplantation.

Aberrant crypts are putative preneoplastic lesions that have been proposed as intermediate biomarkers for colon cancer. The goals of these studies were to determine (i) if the colon cancer chemopreventive agent, sodium phytate, when started 1 week after a single dose of carcinogen, has any effect on the development of aberrant crypt foci (ACF) in treated rats; and (ii) if ACF at an early time period under these conditions correlate with the later formation of tumors in similarly treated animals. The number of ACF with four or more crypts was greater (P = 0.02, Mann-Whitney test) in rats with tumors compared with rats without tumors killed at 36 weeks after the injection of azoxymethane (AOM); the total number of ACF was not significantly different in these two groups. The incidence of tumors in F344 rats treated with AOM without phytate was 83% (10/12) compared to 25% (3/12) in rats treated with AOM plus phytate (P = 0.0045, two-tail Fisher's exact test). The finding of more (P = 0.005, Mann-Whitney test) ACF with four or more crypts in rats without phytate than in rats with phytate at 12 weeks after the injection of AOM is consistent with the hypothesis that the development of larger ACF (with four or more crypts) is predictive of the tumor incidence. These results validate the use of this parameter, i.e. ACF with four or more crypts, as an intermediate biomarker for tumor incidence in this system.

Prostate-specific antigen (PSA), as measured in peripheral blood, is currently the most widely used marker for the assessment of tumor burden in the longitudinal study of patients with carcinoma of the prostate (PCA). Studies from other laboratories have led to the conclusion that a given volume of PCA causes a much higher level of PSA in the peripheral circulation of patients than a similar volume of prostate without carcinoma. We have evaluated PSA in the resected tissues immunohistochemically and in extracts of PCA and of prostates resected because of benign prostatic hyperplasia (BPH) with an enzyme-linked immunosorbent assay. Immunohistochemical results were less quantitative than but consistent with the results of the ELISA of tissue extracts. Immunohistochemically, there was considerable heterogeneity in the expression of PSA by both PCA and BPH both within and among prostatic tissues from different patients. While the levels of expression of PSA in these tissues overlap broadly, PSA is expressed at a lower level in PCA than in BPH when PSA is expressed as a function of wet weight of tissue (p = 0.0095), wet weight of tissue/% epithelium (p less than 0.0001), protein extracted from the tissue (p = 0.0039), or protein extracted/% epithelium (p less than 0.0001).

The development of cancer is characterized by a long latency between the initial exposure to a carcinogenic substance and the final appearance of a macroscopic tumor. Histochemistry has provided the means to identify specific cells involved in this process and many of the changes that take place during the transformation of normal cells to malignant ones. Putative premalignant lesions have been studied extensively in liver carcinogenesis. Histological sections of grossly normal appearing liver a few weeks after treating animals with carcinogen contain discrete areas or “islands” of aberrant enzyme activity that can be detected with enzyme histochemistry and are commonly called “enzyme-altered foci.” These foci display altered activity of one or more enzymes; one of the most common is increased γ-glutamyl transpeptidase (GGT) activity. Since the activities of most enzymes are destroyed by the heat and solvents used for paraffin-embedding, most of this work was done with frozen sections that are often 10 μm in thickness

Gene expression changes during hormonal therapy for prostate cancer reveal candidate diagnostic and drug targets