Beta-catenin expression is altered in human colonic aberrant crypt foci.

The aberrant expression of beta-catenin in colon tumors and the discovery of beta-catenin mutations in small adenomas suggest that alterations of beta-catenin are early events in human colorectal carcinogenesis. Here, we describe the expression of beta-catenin in human aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon. Paraffin-embedded sections of 94 ACF, 12 adenomas, and 10 carcinomas were evaluated for beta-catenin expression by immunohistochemistry. Normal colonic epithelial cells adjacent to these lesions showed strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Cytoplasmic expression of beta-catenin was seen in 25 of 46 ACF with dysplasia and in 2 of 48 ACF with atypia. In ACF with dysplasia, reduced membranous expression of beta-catenin was associated with increased nuclear (P = 0.0013) and cytoplasmic (P = 0.0247) expression. The membranous (P = 0.0003) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0016) and nuclear (P = 0.0266) expressions increased in ACF according to their degree of dysplasia. Likewise, membranous (P = 0.0007) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0050) and nuclear (P = 0.0001) expressions increased from ACF to adenoma to carcinoma. These data suggest that ACF and their aberrant expression of beta-catenin play a role in colon tumorigenesis.

Distinctly different gene structure of KLK4/KLK-L1/prostase/ARM1 compared with other members of the kallikrein family: intracellular localization, alternative cDNA forms, and Regulation by multiple hormones.

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.

Inducible nitric oxide synthase (iNOS) is expressed similarly in multiple aberrant crypt foci and colorectal tumors from the same patients.

Aberrant crypt foci (ACF) are the earliest identified neoplastic lesions in the colon. Aberrant expression of inducible nitric oxide synthase (iNOS or NOS2) has been documented in colorectal tumors, but expression of iNOS has not been reported in human ACF or multiple neoplastic lesions from the same patient. Immunohistochemical expression of iNOS was evaluated in 42 ACF, 14 adenomas, and 25 carcinomas and their adjacent normal mucosa. iNOS was strongly expressed in the normal colonic epithelial cells of all patients; it was markedly reduced in 21 of 42 (50%) ACF and in 14 of 25 (56%) carcinomas. The expression of iNOS was remarkably similar in multiple lesions from the same patient (P < 0.0001). These results suggest that the reduced expression of iNOS is a very early event in the development of some human colorectal tumors, and that host factors control the expression of iNOS similarly in premalignant and malignant colonic epithelial cells.

An efficient procedure for cloning hormone-responsive genes from a specific tissue.

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.

Prostate cancer and other xenografts from cells in peripheral blood of patients.

Good models for the investigation of human prostate cancer are few. Cells from approximately 9.2-21 ml of peripheral blood from patients with metastatic prostate cancer or metastatic colon cancer were injected s.c. into nude mice. Prostate cancer from 2 of 11 patients and colon cancer from 1 of 3 patients were found to be growing as metastases in the lungs of the nude mice. To our knowledge, this is the first report of the formation of xenografts from carcinoma cells taken directly from the peripheral blood of patients. Expanding circulating cancer cells with this approach may have important translational applications including: (a) development of models of human cancers; and (b) sampling of cancers from specific patients for novel molecular and therapeutic approaches.

Loss of fragile histidine triad expression in colorectal carcinomas and premalignant lesions.

Abnormal expression of the fragile histidine triad (FHIT) candidate tumor suppressor gene has been observed in a variety of human tumors, but little is known about its expression during colorectal tumorigenesis. Sections of 70 aberrant crypt foci (ACF), 55 adenomas, 84 primary colorectal carcinomas, and 13 metastatic lesions were evaluated immunohistochemically for Fhit expression. All normal colonic epithelium showed a strong expression of Fhit; 44% of carcinomas showed a marked loss or absence of Fhit expression. The proportion of carcinomas with reduced expression showed an increasing trend (a) with decreasing differentiation and (b) in tumors with metastases (62%) compared with tumors without metastases (38%). The proportion of metastatic lesions (12 of 13) with reduced expression of Fhit was even greater. Although only a small proportion of ACF and adenomas showed a reduction of Fhit expression, the reduced expression of Fhit was strongly associated with the degree of dysplasia in both ACF (P = 0.0002) and adenomas (P = 0.0085). The findings of reduced expression of Fhit in a small proportion of colonic precancerous lesions and in increased proportions of primary and metastatic colorectal cancers suggest that Fhit plays a role in the development and progression of some colon carcinomas.

Full-length cDNA sequence and genomic organization of human NKX3A - alternative forms and regulation by both androgens and estrogens.

NKX3A (NKX3.1) is a recently identified androgen-regulated gene that is largely specific to prostate for expression and likely to code for a homeobox protein. Here we report the full-length mRNA and genomic organization of human NKX3.1. There are at least five different splice variants of NKX3.1 mRNA that result in different open reading frames (ORFs). There is extensive similarity between the human and the mouse NKX3.1 cDNA sequences outside of the ORFs (greater than 60% overall identity), which may be involved in modulating NKX3.1 expression. In addition to its androgen regulation in the prostate cancer cell line LNCaP, we show that NKX3.1 expression is androgen-dependent in the CWR22 prostate cancer xenograft model. Interestingly, NKX3.1 is highly expressed in the androgen-independent derivative CWR22R in the absence of androgens, indicating that it may be deregulated in advanced prostate cancer. Using a Green Fluorescent Protein fusion construct, we show that NKX3.1 is a nuclear protein consistent with its proposed function as a homeobox transcription factor. Furthermore, in addition to androgens, NKX3.1 expression is up-regulated by 17beta-estradiol, but not by progesterone, dexamethasone, or 3,5,3'-triiodothyronine in LNCaP cells. Regulation of NKX3.1 by androgens and 17beta-estradiol in prostate cancer cells and its deregulation in androgen-independent prostate cancer suggest that it may have important regulatory roles during prostate cancer progression.

Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays.

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 overexpressed 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.

Molecular cytogenetic studies of a serially transplanted primary prostatic carcinoma xenograft (CWR22) and four relapsed tumors.

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 alpha-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.

A new human prostate carcinoma cell line, 22Rv1.

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.

Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.

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.

Changes in cyclin dependent kinase inhibitors p21 and p27 during the castration induced regression of the CWR22 model of prostatic adenocarcinoma.

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 that expressed p27 increased from 25+/-3% in intact controls to 51+/-8% at 3 days post-castration and to 80 to 95% at days 7, 21, 28 and 42 days post-castration. Testosterone treatment from 21 to 28 days post-castration resulted in an increase in Ki-67 antigen positive cells to 200% of intact controls and a concomitant reduction in p27 expressing cells to about 50% of intact controls. Castration-induced changes in p27 expression were not observed in the CWR22R tumor, a transplantable relapsed derivative of the CWR22 tumor. CONCLUSION: These findings suggest that p27 expression is regulated negatively by androgens and that increased expression of p27 in CWR22 xenografts may be involved in the suppression of proliferation following castration.

Overexpression of cyclin D1 is rare in human prostate carcinoma.

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 transcripts. 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.

The identification of monoclonality in human aberrant crypt foci.

Malignant neoplasms, including colon cancers, are thought to arise from a single initiated progenitor cell. Aberrant crypt foci (ACF) are putative precursors of at least some colon cancers. The pattern of X chromosomal inactivation, which is identified by the differential methylation of a site near a polymorphic CAG repeat in the androgen receptor gene, was used to determine the clonality status of 11 ACF from eight female patients. Ten of 11 ACF were found to be monoclonal aberrations. The eleventh ACF appeared monoclonal, but nonrandom inactivation of the X chromosome was also seen in normal crypts from this patient. These results clearly demonstrate that: (a) a high percentage of ACF lesions are neoplastic rather than hyperplastic; and (b) ACF are the earliest identified neoplastic lesions in the colon.

Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.

The human prostate cancer (CaP) xenograft, CWR22, mimics human CaP. CWR22 grows in testosterone-stimulated nude mice, regresses after castration, and recurs after 5-6 months in the absence of testicular androgen. Like human CaP that recurs during androgen deprivation therapy, the recurrent CWR22 expresses high levels of androgen receptor (AR). Immunohistochemical, Western, and Northern blot analyses demonstrated that AR expression in the androgen-independent CWR22 is similar to AR expression in the androgen-dependent CWR22 prior to castration. Expression of prostate-specific antigen and human kallikrein-2 mRNAs, two well-characterized androgen-regulated genes in human CaP, was androgen dependent in CWR22. Despite the absence of testicular androgen, prostate-specific antigen and human kallikrein-2 mRNA levels in recurrent CWR22 were higher than the levels in regressing CWR22 tumors from 12-day castrate mice and similar to those in the androgen-stimulated CWR22. Other AR-regulated genes followed a similar pattern of expression. Differential expression screening identified androgen regulation of alpha-enolase and alpha-tubulin as well as other unknown mRNAs. Insulin-like growth factor binding protein-5, the homeobox gene Nkx 3.1, the AR coactivator ARA-70, and cell cycle genes Cdk1 and Cdk2 were androgen regulated in CWR22. In recurrent CWR22, the steady-state levels of all these AR-dependent mRNAs were similar to those in the androgen-stimulated CWR22, despite the absence of testicular androgen. Expression of AR and AR-regulated genes in the androgen-deprived recurrent CWR22 at levels similar to the androgen-stimulated CWR22 suggests that AR is transcriptionally active in recurrent CWR22. Induction of these AR-regulated genes may enhance cellular proliferation in relative androgen absence but through an AR-dependent mechanism. Alternatively, in androgen-independent tumors, induced expression of the AR-regulated gene network might result from a non-AR transcription control mechanism common to these genes.

Providing human tissues for research: how to establish a program.

The Cooperative Human Tissue Network is a group composed of cooperating academic institutions that supply human tissues to researchers studying a wide range of neoplastic and other diseases. The experience of the Cooperative Human Tissue Network in establishing methods of prospective tissue collection and in developing tumor banks is discussed to aid institutions in establishing tissue resources for their local investigators, who may wish to use human tissues in current or future research projects. The advantages to pathology departments and to associated medical institutions of establishing an organized tissue resource include ensuring proper institutional review board approval of research projects using human tissues, protecting diagnostic specimens, creating new opportunities for extramural research, increasing the speed of diagnostic specimen transport to surgical pathology, and providing educational and research opportunities for pathologists and pathology residents. Methods of tissue collection, processing, storage, data collection, and supply are outlined. Also, resources necessary to begin organized tissue collection, including personnel, space, equipment, and supplies, are discussed.

Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3'-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells.

Etk/Bmx is the newest member of Btk tyrosine kinase family that contains a pleckstrin homology domain, an src homology 3 domain, an src homology 2 domain, and a catalytic domain. Unlike other members of the Btk family kinases, which are mostly hemopoietic cell-specific, Etk/Bmx is preferentially expressed in epithelial and endothelial cells. We first identified this kinase in prostate cancer [Robinson, D., He, F., Pretlow, T. & Kung, H. J. (1996) Proc. Natl. Acad. Sci. USA 93, 5958-5962). Here we report that Etk is engaged in phosphatidylinositol 3-kinase (PI3-kinase) pathway and plays a pivotal role in interleukin 6 (IL-6) signaling in a prostate cancer cell line, LNCaP. Our evidence that PI3-kinase is involved in Etk activation includes: (i) Wortmannin, a specific inhibitor of PI3-kinase, abolished the activation of Etk by IL-6; (ii) a constitutively active p110 subunit of PI3-kinase was able to activate Etk in the absence of IL-6; and (iii) a dominant negative p85 subunit of PI3-kinase mutant blocked the activation of Etk by IL-6. Interestingly, IL-6 treatment of LNCaP induced a remarkable neuroendocrine-like differentiation phenotype, with neurite extension and enhanced expression of neuronal markers. This phenotype could be abrogated by the overexpression of a dominant-negative Etk, indicating Etk is required for this differentiation process.

Putative preneoplastic changes identified by enzyme histochemical and immunohistochemical techniques.

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.