ABSTRACT Prostate cancer tends to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy. We used two variants of the human prostate cancer

cell line LNCaP to study gene expression differences during prostate cancer progression to androgen-independent disease. Production of prostate-specific antigen was regarded as a marker of

androgen-dependence and loss of prostate-specific antigen was regarded as a marker of androgen-independence. mRNA from both cell lines was used for cDNA microarray screening. Differential

expression of several genes was confirmed by Northern blotting. Monoamine oxidase A, an Expressed Sequence Tag (EST) similar to rat P044, and EST AA412049 were highly overexpressed in

PC-3 cells. The EST similar to rat P044, the EST similar to galectin-1, follistatin, annexin I, and the interferon-inducible gene 1–8U were also expressed in benign prostatic hyperplasia

tissue. The Y-linked ribosomal protein S4, Mat-8, and EST AA307912 were highly expressed in benign prostatic hyperplasia tissue. Additionally, both confirmation of differential expression in

Northern blots and _in situ_ hybridization were carried out for monoamine oxidase A, the EST similar to rat P044, the EST similar to galectin-1, fatty acid-binding protein 5, and the

interferon-inducible gene 1–8U. We identified several potential prostate cancer markers, indicating that the method used is a useful tool for the screening of cancer markers, but other

methods, such as _in situ_ hybridization, are needed to further investigate the observations. SIMILAR CONTENT BEING VIEWED BY OTHERS MED19 ENCODES TWO UNIQUE PROTEIN ISOFORMS THAT CONFER

PROSTATE CANCER GROWTH UNDER LOW ANDROGEN THROUGH DISTINCT GENE EXPRESSION PROGRAMS Article Open access 25 October 2023 CHARACTERISATION OF CELL LINES DERIVED FROM PROSTATE CANCER PATIENTS

WITH LOCALISED DISEASE Article Open access 01 June 2023 PROSTATE EPITHELIAL GENES DEFINE THERAPY-RELEVANT PROSTATE CANCER MOLECULAR SUBTYPE Article Open access 26 April 2021 INTRODUCTION

Prostate cancer is the most commonly diagnosed cancer among men in Western industrialized countries. The growth of prostate cancer is usually androgen-dependent at the beginning, but tends

to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy (Schroder, 1998). The transition from hormone-dependent to hormone-independent

tumorigenesis is believed to be a consequence of multiple genetic alterations. This multistep process is thought to include a cascade of genetic alterations caused by activation of oncogenes

and/or inactivation of tumor suppressor genes (Fearon and Vogelstein, 1990; Knudson, 1993). A widely used _in vitro_ model of prostate cancer is the LNCaP cell line (Horoszewicz et al,

1980). Androgens are essential for growth of LNCaP cells, unless they have become transformed to androgen-independent clones (van Steenbrugge et al, 1991). Androgen-dependent and

androgen-independent LNCaP cell lines have previously been used for studying differentially expressed genes in the respective stages of prostate carcinoma. B cell translocation gene 1, the

UDP glucuronosyl transferase gene 2B15, and two unknown genes are differentially expressed (Chang et al, 1997). The cell lines used expressed both prostate-specific antigen (PSA) and

prostatic acid phosphatase and contained androgen receptors (although one cell line was androgen-independent). Differential expression of fibronectin, E2 ubiquitin-conjugating enzyme,

metalloproteinase-related collagenase, and breast basic conserved gene was shown in another androgen-independent LNCaP cell line in a study by Stubbs et al, 1999. Recently, differential

display analysis was successfully used for identification of a cDNA (DD3) with prostate cancer-specific expression (Bussemakers et al, 1999). To study gene expression changes during prostate

cancer progression, we used two LNCaP cell line variants. Loss of PSA production in LNCaP cells was regarded as a marker of androgen-independent growth of the cells. The non–PSA-producing

LNCaP cell line (LNCaP−) served as a model of progressive prostate cancer, whereas the PSA-producing LNCaP cell line (LNCaP+) served as a model of well-differentiated prostate cancer. The

aim of this study was to identify differentially expressed genes in LNCaP+/− cells using cDNA array technology. With this technology, the expression of several thousand genes can be observed

in single hybridization screening. Thus, this technology is a powerful tool in cancer research with potential for the identification of new cancer markers. Several genes with prominent

expression in one of the cell line variants were analyzed by Northern blot hybridization in the LNCaP+/−, PC-3, and DU-145 cell lines and in benign prostatic hyperplasia (BPH) tissue, and

some promising markers were investigated by _in situ_ hybridization (ISH). RESULTS OVERVIEW OF THE SCREENING RESULTS In gene expression microarray analysis, 7075 human cDNAs were screened.

Among genes expressed at high levels in both LNCaP cell lines there were several genes known to take part in the regulation of cell growth, apoptosis, or oncogene action. These genes are

presented in Table 1. Many genes were expressed at low level: signal values below 200 (range: 76 to 199) were detected for 2013 genes. Of these genes, 1252 (62%) were EST (Expressed Sequence

Tags) with no homology to known sequences; 207 (10%) were EST with similarity to some known sequences; and 554 (28%) were known clones different from EST. DIFFERENTIALLY EXPRESSED GENES The

genes expressed most prominently in LNCaP+ and LNCaP− cells are presented in Tables 2 and 3, respectively. Androgen receptor was expressed in LNCaP+ cells with a signal value of 2941, 7.9

times overexpressed in these cells (Table 2). The respective value in LNCaP− cells was 373, indicating the presence of androgen receptor transcripts in these cells as well. In LNCaP+ cells,

the genes for four serine proteases, PSA (Accession number (AC): M24543), prostate-specific glandular kallikrein (hK2, AC: S39329), TMPRSS2 (AC: U75329), and kallikrein 1 (AC: M12706), were

among the thirty most overexpressed genes, whereas in LNCaP− cells there was only one serine protease, trypsin 3 (AC: X71345), among the ninety most overexpressed genes. Furthermore, genes

for three transmembrane proteins, TMPRSS2, Mat-8 (AC: AA826766) and nma (AC: U23070), were among the ninety most overexpressed genes in LNCaP+ cells. In LNCaP− cells, there were several

interferon-inducible genes that were overexpressed. Among the ninety most overexpressed genes were cDNAs for 2′-5′ oligoadenylate synthetase 2 (OAS2, AC: M87284), MxA (AC: M30817),

interferon-inducible protein 17 (AC: AA428847), a 56 kDa protein (AC: X03557), interferon-α–inducible protein 27 (AC: AA302123), interferon-γ–inducible protein 16 (AC: S75433), MxB (AC:

M33883), and the interferon-inducible gene 1–8U (AC: X57352). Protein phosphatase 3 and myosin VI were differentially expressed in the whole array, but they were expressed at comparable

levels in both LNCaP+ and LNCaP− cells according to the results of Northern blotting (data not shown). ANALYSIS OF OVEREXPRESSED GENES IN LNCAP+ CELLS The Y-linked ribosomal protein S4 was

expressed strongly in BPH tissue, was expressed at lower levels in LNCaP+, and was hardly detectable in PC-3 or LNCaP− cells (Table 4, Fig. 1a). Monoamine oxidase A (MAOA) was expressed in

LNCaP+ cells and at lower levels in BPH tissue and LNCaP− cells (Table 4, Fig. 1a), and was not seen in PC-3 cells. ISH showed the localization of MAOA transcripts in both benign and

malignant epithelium (Fig. 2, a and b). Mat-8 was expressed in BPH tissue and in LNCaP+ cells, whereas transcripts encoding Mat-8 were absent in PC-3 and LNCaP− cells (Table 4, Fig. 1a). An

mRNA for an EST similar to rat P044 was most prominently expressed in LNCaP+ cells and BPH tissue, and was weakly expressed in LNCaP− cells (Table 4, Fig. 1a). It was expressed at a low

level in ISH, but was detectable in benign and malignant epithelia (Fig. 2, c and d). ISH showed that EST AA781244 was expressed at similar levels in benign and malignant epithelia (Fig. 2,

e and f). EST AA781244 was weakly expressed in LNCaP+ cells and BPH tissue in Northern blots (Table 4). The gene encoding EST AA412049 was expressed only in LNCaP+ cells in Northern blots

(Table 4), but in ISH it was detectable in both benign and malignant epithelia (Fig. 2, g and h). ANALYSIS OF OVEREXPRESSED GENES IN LNCAP− CELLS In the prostate cell lines studied,

tissue-type plasminogen activator (tPA) was expressed only in LNCaP− cells; no expression was seen in BPH tissue (Table 4, Fig. 1b). MxB, like tPA, was expressed only in LNCaP− cells (Table

4, Fig. 1a). Expression of EST AA307912 was detected in the androgen-independent cell lines, DU-145 and PC-3, as well as in LNCaP− cells (Fig. 1b). However, EST AA307912 was also expressed

in BPH tissue (Table 4). The EST similar to galectin-1 was expressed most strongly in LNCaP− cells (Table 4, Fig. 1b), otherwise, its expression pattern was comparable to that of EST

AA307912. In previous studies, galectin-1 expression was observed only in androgen-independent DU-145, PC-3, and metastatic PC-3M cell lines (Ellerhorst et al, 1999a), similar to the

expression pattern of the EST similar to galectin-1. In prostate tissue, the EST similar to galectin-1 was expressed in the stroma (Fig. 2, i and j), where the expression of galectin-1 has

also been localized by immunohistochemistry (Ellerhorst et al, 1999b). Follistatin and fatty acid-binding protein 5 (Table 4, Fig. 1a) were expressed in LNCaP− cells and in the

androgen-independent PC-3 cell line. Several mRNA species for follistatin were detected in LNCaP− cells (Fig. 1a) and one was also expressed in BPH tissue. Fatty acid-binding protein 5 was

expressed in both benign and malignant epithelium by ISH (Fig. 2, k and l), although it was not expressed in BPH tissue in Northern blot. EST AA609749 was most abundantly expressed in LNCaP−

cells, weakly expressed in PC-3 cells and BPH tissue, whereas the gene encoding this EST was silent in LNCaP+ cells (Table 4, Fig. 1c). Annexin I (Fig. 1c) and 1–8U (Fig. 1a) were expressed

in both LNCaP− cells and BPH tissue (Table 4). Annexin I was also detectable in PC-3 cells. According to the results of ISH, the expression of 1–8U was interesting (Fig. 3, a to e) because

it was concentrated in some cells of cancer areas. In benign tissue, 1–8U was mainly localized in epithelial cells. However, in some areas, expression was also detected in the stroma.

Phospholipase D1 was strongly expressed in LNCaP− cells and low amounts of mRNA were detected in PC-3 cells, whereas LNCaP+ cells and BPH tissue totally lacked expression (Table 4, Fig. 1c).

According to the cDNA expression array analysis, OAS2 was 34.6 times overexpressed in LNCaP− cells (Table 3). In Northern blot (Fig. 1d), OAS2 transcripts were clearly observed only in

LNCaP− cells, not in BPH tissue, DU-145, or PC-3 cell lines. In ISH, expression of OAS2 (Fig. 3, g and h) was detected in some prostate cancer areas in the samples analyzed. Expression was

also seen in non-malignant prostate tissue, but the sparsely distributed transcript level was low. DISCUSSION Here we describe the results of cDNA microarray screening of two LNCaP cell line

variants. Sixteen genes were selected from the array; differential expression of fourteen of these genes was confirmed by Northern blotting. Among differentially expressed genes, there are

many potential prostate cancer markers. Several genes studied more closely here have previously been investigated with regards to their overall association with cancer. These genes are tPA,

OAS, follistatin, annexin I, 1–8U, PSA, hK2, TMPRSS2, MAOA, and Mat-8. Of these, only PSA, hK2, TMPRSS2, tPA, and follistatin have been studied specifically in prostate cancer. Additionally,

six EST clones or clones without a known product or function were found to be differentially expressed (EST similar to rat P044, AC: AI192351; EST similar to galectin-1, AC: AA340061; AC:

AA781244; AC: AA412049; AC: AA307912; and AC: AA609749). Our results indicate that the well known androgen-regulated prostatic proteins PSA and hK2 are highly expressed in LNCaP+ cells,

whereas they are down-regulated in LNCaP− cells. The galectins are a family of lectins that probably mediate cell adhesion, regulate cell growth, and trigger or inhibit apoptosis. The

expression pattern of some galectins is altered in breast, colon, prostate, and thyroid carcinomas (Perillo et al, 1998). Our finding of overexpression of an EST similar to galectin-1 in

LNCaP− cells suggests that these cells are transformed to a more progressive stage of prostate cancer compared with galectin-1, which was expressed in DU-145, PC-3, and PC-3M cells but not

in LNCaP cells analyzed by Northern blotting and immunoblotting (Ellerhorst et al, 1999a). Furthermore, another protein sharing similarity with the galectin family, PCTA-1, is expressed in

prostate cancer tissue (Su et al, 1996). Overexpression of follistatin in LNCaP− cells is in accordance with the theory of follistatin overproduction favoring androgen-independent prostate

cancer growth (Thomas et al, 1997). Activin inhibits the growth of DU-145 and LNCaP cells, even after androgen-stimulation (Wang et al, 1999), but not PC-3 cell lines. This inhibition may be

prevented by follistatins (Thomas et al, 1997). An interesting question is how to overcome the growth-promoting influence of follistatin in prostate cancer cells. MxA and MxB belong to the

family of large GTPases. Cytoplasmic human MxA protein mediates resistance to multiple RNA viruses, whereas no antiviral activity has been found for human MxB protein (Melen et al, 1996).

The genes for MxA and MxB are located in the same chromosomal area as the TMPRSS2 gene, 21q22.3 (Paoloni-Giacobino et al, 1997). Interestingly, MxA and MxB were up-regulated in the

androgen-independent cell line (LNCaP−), whereas TMPRSS2 was down-regulated in these cells. Thus, deletion of this chromosomal area is not likely to clarify the differential expression of

these genes in the cell line variants, and up- or down-regulation is due to specific regulatory factors. Additionally, expression of TMPRSS2 is androgen-regulated (Lin et al, 1999; Vaarala

et al, unpublished data) and low amounts of TMPRSS2 as well as other androgen-dependent serine proteases in LNCaP− cells reflect a change in the hormonal status/response of this cell line

variant. The antitumor effectiveness of interferons _in vivo_ and _in vitro_ is well documented. OAS is an interferon-inducible enzyme that polymerizes 2′-5′ oligoadenylates from adenosine

triphosphate. 2′-5′ oligoadenylates, in turn, are responsible for RNaseL activation, which is needed for RNA degradation and antiviral action (Stryer, 1988). However, only some of the

interferon-inducible genes activated in LNCaP− cells are known to belong to the OAS-RNaseL pathway. The uneven distribution of OAS2 mRNA in prostate samples found here may reflect

participation in cell growth regulation or an as yet unknown process involved in the development of cancer cell populations with different endocrine characteristics. The cytocidal effect of

treating the HT29 human colon carcinoma cell line with both TNF-α and interferon-γ is reported to be accompanied by an increase in OAS levels and apparent rRNA breakdown (Chapekar and

Glazer, 1988). However, the antiproliferative effects of interferons did not correlate with induction of OAS in human lung cancer (Martyre et al, 1988). The gene encoding 1–8U has been

observed to be strongly expressed in ulcerative colitis-associated colon cancers (Hisamatsu et al, 1999). This gene is highly interferon-inducible, but its function is unclear. There is some

evidence of a link between prostate cancer and colon cancer (Moore et al, 1998) and, furthermore, the expression of certain genes is similarly altered in cancers of different tissues (eg,

KAI1). It is therefore possible that 1–8U also has some association with prostate cancer. The activation of interferon-inducible genes in LNCaP− cells seems to be involved in the transition

to more aggressive growth or malignant transformation, as in the case of 1–8U in colon cancer (Hisamatsu et al, 1999). The possibility of this induction being a result of _mycoplasma_

contamination was ruled out by regular testing of the cells. Mat-8 is an 8-kd transmembrane protein homologous to phospholemman (Morrison et al, 1995). It is differentially expressed in the

MCF-7 breast cancer cell line and in a progressive variant of MCF-7 (Schiemann et al, 1998). The kallikrein gene family and Mat-8 genes are located in chromosomal region 19q, suggesting that

this genetic area could be vulnerable to regulatory changes during transformation. The Y-linked ribosomal protein S4 was most prominently expressed in BPH tissue and was also detectable in

LNCaP+ cells. The gene encoding this protein is overexpressed in a non-metastatic variant of rat rhabdomyosarcoma, SMF-Da (Daigneault et al, 1995). The expression of several other ribosomal

proteins is also altered in prostate cancer and prostate cancer cell lines (Vaarala et al, 1998). Angiostatin, an inhibitor of angiogenesis and growth of tumor metastases, is generated by

urokinase-mediated proteolysis of plasminogen in the PC-3 cell line (Gately et al, 1997). Recently, PSA has also been shown to have antiangiogenic activity (Fortier et al, 1999). Another

plasminogen activator, tPA, was overexpressed in LNCaP− cells. Down-regulation of PSA production possibly leads to accumulation of plasminogen in these cells, which might then promote

up-regulation of the gene encoding tPA. However, according to Gately et al, 1997, tPA seems not to have similar activity to urokinase and PSA (Fortier et al, 1999), and the concentration of

tPA is not altered in prostate cancer tissue compared with normal prostate tissue (Plas et al, 1998). Unfortunately, cDNA for urokinase was not available in the array. The plasminogen gene

was expressed at a low level with signal values of 575 and 281 in LNCaP+ and LNCaP− cells, respectively. Several electronic databanks now offer tissue- or cell line-specific expression data

for cDNA clones. A sophisticated model for expression studies in prostate tissue specimens, using the microarray technique, has been presented recently (Cole et al, 1999). Information given

by cDNA arrays, as in the present study, is a valuable tool in cancer research, because genes with prominent or very low expression can be compared with expression data available on another

disease states. For example, finding a gene with high expression in LNCaP+ and LNCaP− cells, with no or low expression in BPH or normal prostate tissue, would reveal a potential new prostate

cancer marker. Careful examination of clones is still needed, because there are differences within and between LNCaP cell lines and prostate cancer, as indicated by ISH analyses. MATERIALS

AND METHODS CELLS AND TISSUES LNCaP-FGC, PC-3, and DU-145 cells were purchased from ATCC (Rockville, Maryland). Production of PSA by LNCaP cells was followed by using DELFIA PSA kits

(Wallac, Turku, Finland); cell clone variants with high PSA production and undetectable PSA production were selected. Lack of _mycoplasma_ contamination was confirmed regularly (Gen-Probe,

San Diego, California). Tissues for ISH were taken from prostatic adenocarcinoma patients treated by radical prostatectomy or transurethral electroresection of the prostate. BPH tissue was

obtained from patients treated by radical/total prostatectomy. These treatments are widely used and accepted in cases of BPH and prostate cancer. Tissues were removed only for treatment

purposes and were used for this study only after diagnostic confirmation by histopathology. RNA ISOLATION, CDNA MICROARRAY, AND CLONE SEQUENCING Total RNA was isolated from the cells using

TRIzol (Life Technologies, Gaithersburg, Maryland). Poly-A RNA was isolated using a QuickPrep Micro mRNA Purification Kit (AmershamPharmacia, Uppsala, Sweden). mRNA was used for Human UniGEM

V v1.0 Custom Screening (GenomeSystems Inc., St. Louis, Missouri). mRNAs from LNCaP+/− cells were labeled with different fluorescent labels. Average signals for the elements in the array

were achieved for both probes. For details of screening and clone selection, see http://www.genomesystems.com. Results are presented as either probe average signal values (arbitrary units)

or as overexpression, when signal values of a cell line are divided by signal values in the other cell line. Histologically confirmed tissue samples of BPH were used for the isolation of

total RNA by the CsCl gradient method. Poly-A RNA was isolated using oligo(dT)-cellulose (AmershamPharmacia) and standard methods. Clones selected according to screening were purchased from

GenomeSystems Inc. and were further sequenced using an ABI377 automatic sequencer (Applied Biosystems, Branchburg, New Jersey) to confirm the sequence data and clone orientation. NORTHERN

BLOT ANALYSIS mRNA isolated from the cells and tissues were loaded on the gel. Northern blots were carried out as recommended by the membrane manufacturer (Boehringer Mannheim, Mannheim,

Germany). Hybridization was carried out with 32P-labeled cDNA and GAPD in plasmid vectors. The filters were washed under high stringency conditions and exposed to Kodak MS film (Eastman

Kodak, Rochester, New York) with intensifying screens. The filters were stripped and removal of probes was confirmed by exposure of the blots before each hybridization. _IN SITU_

HYBRIDIZATION Antisense and sense [α-35S]CTP-labeled RNA probes were transcribed with T7 or SP6 RNA polymerases using linearized plasmids as templates. The specific activities of the

synthesized RNA probes were approximately 6 × 106 cpm/μl. ISH was carried out as previously described (Mustonen et al, 1998) using tissue samples obtained from transurethral electroresection

of prostate cancer patients and tissue blocks containing BPH tissue and prostate cancer tissue. The hybridization temperature was 60° C. The slides were stained with hematoxylin and eosin,

and mounted with Pertex (Histolab, Göteborg, Sweden). Each probe was analyzed in a minimum of two BPH tissue samples and two prostate cancer samples. ACCESSION CODES ACCESSIONS

GENBANK/EMBL/DDBJ * AA302123 * AA307912 * AA340061 * AA399275 * AA412049 * AA428847 * AA504039 * AA641953 * AA781244 * AA826766 * AA888264 * AB002387 * AB006987 * AC002550 * AF007111 *

AF015450 * AF032886 * AF035283 * AF036268 * AF052578 * AF070539 * AF102803 * AI352370 * AI422824 * AI536671 * AI631255 * AI720570 * AI809937 * AI819274 * AI92351 * AI929331 * AL034562 *

D01096 * D10656 * D29810 * D84488 * D86042 * J04469 * L02911 * L25610 * M12706 * M16768 * M19481 * M24543 * M26602 * M27430 * M30817 * M33883 * M34181 * M60047 * M62397 * M64110 * M69226 *

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U84214 * U90543 * U95742 * V00568 * X03484 * X03557 * X05908 * X57352 * X59656 * X61587 * X67951 * X68148 * X71345 * X75593 * X79201 * Y07848 REFERENCES * Bussemakers MJ, van Bokhoven A,

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Ms. Helmi Konola and Ms. Mirja Mäkeläinen for technical assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Biocenter Oulu, World Health Organization Collaborating Centre for Research

on Reproductive Health, University of Oulu, Oulu, Finland Markku H Vaarala, Katja Porvari & Pirkko Vihko * Department of Pathology, University of Oulu, Oulu, Finland Atte Kyllönen *

Department of Biosciences, Division of Biochemistry, World Health Organization Collaborating Centre for Research on Reproductive Health, University of Helsinki, Helsinki, Finland Pirkko

Vihko Authors * Markku H Vaarala View author publications You can also search for this author inPubMed Google Scholar * Katja Porvari View author publications You can also search for this

author inPubMed Google Scholar * Atte Kyllönen View author publications You can also search for this author inPubMed Google Scholar * Pirkko Vihko View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Pirkko Vihko. ADDITIONAL INFORMATION Grants provided by Research Council for Health, Academy of Finland

(3314 and 40990); and the Ministries of Education, Social Affairs and Health, and Foreign Affairs, Finland; Finnish Cancer Foundation. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Vaarala, M., Porvari, K., Kyllönen, A. _et al._ Differentially Expressed Genes in Two LNCaP Prostate Cancer Cell Lines Reflecting Changes during Prostate

Cancer Progression. _Lab Invest_ 80, 1259–1268 (2000). https://doi.org/10.1038/labinvest.3780134 Download citation * Received: 20 March 2000 * Published: 01 August 2000 * Issue Date: 01

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