Lipofection-mediated immortalization of human prostatic epithelial cells of normal and malignant origin using human papillomavirus type 18 DNA.

Human papillomavirus (HPV) type 18 DNA was introduced into epithelial cell strains derived from normal and cancer tissues of human prostatectomy specimens by the lipofection transfection method. Two cell lines were established: PZ-HPV-7 (transfected normal cell) and CA-HPV-10 (transfected cancer-derived cell). These lines have been maintained for over 100 passages. Incorporation of HPV type 18 DNA was confirmed by polymerase chain reaction. Immunocytochemical analysis showed expression of keratins 5 and 8, similar to the cells of origin, and the early region 6 oncoprotein of HPV. PZ-HPV-7, derived from normal diploid cells, had a modal chromosome number of 46 in early passages but became tetraploid later. CA-HPV-10 cells were aneuploid, and some retained the double minute chromosomes that were noted in the cancer-derived cells of origin. The cell lines showed a typical transformed morphology and were nontumorigenic in nude mice. We conclude that human prostatic epithelial cells derived from both normal and cancer tissues have been successfully transformed to immortality with HPV type 18 DNA. The establishment of these cell lines provides an opportunity for further development of an in vitro model of carcinogenesis for prostate cancer.

Loss of heterozygosity of chromosome 8 microsatellite loci implicates a candidate tumor suppressor gene between the loci D8S87 and D8S133 in human prostate cancer.

To search for specific chromosome 8 aberrations in human prostate cancer, DNA was isolated from 44 human prostate tumor samples. Twenty six tumor samples were obtained from locally progressive tumors by transurethral resection, 12 were from radical prostatectomy specimens, and 6 were from lymph node metastases. Tumor DNAs were screened for allelic losses using 16 highly polymorphic microsatellite loci (14 covering the p arm, 2 on the q arm). In general, the detected deletions were large. In 59% of the tumor DNAs, allelic loss of 3 or more 8p loci was observed. Loss of 8p loci occurred in between 36 and 69% of the informative cases; for the two 8q markers, the percentages of loss were 11 and 25%, respectively, indicating preferential loss of (part of) 8p. In one tumor, two separate 8p deletions were found. The percentage of loss of heterozygosity was considerably higher in transurethral resection (65%) and lymph node metastases (83%) than in radical prostatectomy specimens (33%), suggesting that 8p deletion is a relatively late step in tumor progression. The maximal overlapping deleted region in all tumor DNAs is between the distal locus D8S133 and the proximal locus D8S87, indicating the localization of a candidate tumor suppressor gene within this region.

Gain and loss of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate cancers.

Fluorescence in situ hybridization (FISH) with centromere probes was used to investigate numerical aberrations of chromosomes 1, 7, 8, 10, 18, and Y in 46 prostate carcinoma (PC) and 11 benign prostatic hyperplasia (BPH) samples. None of the benign specimens showed any chromosomal aberration. Forty-one of 46 PC specimens showed numerical aberrations of one or more chromosomes. All investigated chromosomes showed numerical aberrations in at least 30% of the specimens, gain being more frequent than loss. Comparison of DNA flow cytometry (FCM) and FISH results showed that not only aneuploid tumors but also most diploid tumors harbored numerical chromosome aberrations. Chromosome 10 was the most frequently gained (65%), and Y the most frequently lost chromosome (14%). Nonmetastatic and metastatic tumors differed significantly (P < .05) in the number of copies for chromosomes 7, 8, and 10, but not for 1, 18, and Y. These results suggest strongly that gains of chromosomes 7, 8, and 10 are involved in PC progression.

Cytogenetic characterization of an established xenografted prostatic adenocarcinoma cell line (PC-82).

Detailed cytogenetic analysis was performed of a xenografted human prostatic adenocarcinoma cell line PC-82. A direct preparation method was developed that yielded metaphases of good quality. Flow cytometric data and banding analysis of metaphases showed a near-tetraploid karyotype with 18 consistent marker chromosomes. As a result of the rearrangements involved, parts of chromosomes 2, 3, 4, 7, 9, 10, 15, 18, and 21 were homozygous, while regions on 2p, 13q, and 17q were apparently completely lost. In contrast to this, some regions on #2, #5, and, especially, on #1 were present in three or even four times the normal copy number. Comparison of affected chromosomes in PC-82 with all data available on prostatic carcinoma showed chromosomes 1, 2, 3, 6, 7, 10, and 15 to be involved in rearrangements in over 50% of all prostatic carcinomas. When only data from primary prostatic adenocarcinomas (including PC-82) were taken into account it appeared that chromosomes 1, 7, and 10 were involved in all five primary tumors studied.

Cytogenetic analysis of 39 prostate carcinomas and evaluation of short-term tissue culture techniques.

Karyotypic analysis was performed on 102 prostate cancer specimens which were obtained through radical prostatectomy, transurethral resection, or regional lymph node dissection. Short term tissue culture was applied in all cases. Of the media and growth factors evaluated, F12/DMEM, supplemented with 2% fetal calf serum, insulin, epidermal growth factor, hydrocortisone, and cholera toxin produced the largest increase of in vitro proliferation. Such in vitro cultured cells were all phenotypically acinar epithelial cells, the supposed targets for neoplastic transformation. Stromal cell growth appeared to be completely suppressed. Of the three culture techniques investigated, the method developed in Lund, Sweden, was the most successful: 11/15 cultures yielded metaphases and, in three of these, clonal aberrations were identified. All 39 karyotypes obtained essentially had a 46,XY karyotype with clonal aberrations (eight cases) and/or nonclonal aberrations (30 cases). Clonal structural aberrations involved 2p, 3q, 11p, 17p, and 21q. The clonal numerical aberrations found were: + 8, + dmin, and -Y. The most frequently observed nonclonal aberrations were 8p deletions (five cases) and loss of 6, 7, 8, 15, 17, 18, 21, and/or Y (> or = five cases). In summary, clonal aberrations were observed in 20% of the evaluable PC cell cultures, and nonclonal aberrations in 77%. So, although diploid cells without clonal abnormalities still had a growth advantage, under optimal conditions PC cells were able to proliferate in primary in vitro culture.

Specific cytogenetic aberrations in two novel human prostatic cell lines immortalized by human papillomavirus type 18 DNA.

Using chromosome banding and fluorescence in situ hybridization (FISH) with painting probes, sequential cytogenetic analysis was performed of two novel prostate cell lines, PZ-HPV-7 and CA-HPV-10, established by human papillomavirus (HPV) 18 DNA transformation. PZ-HPV-7 originates from a normal diploid prostate epithelial cell strain. PZ-HPV-7 progressed from an initial diploid to a hypertetraploid chromosome number with a relative gain of chromosomes 5 and 20 (7 to 8 copies each). Structural changes were limited; 3p- (2 copies), 3q- (1 copy), and possibly a der(16p;12q). CA-HPV-10 originates from an epithelial cell strain derived from a high-grade human prostate cancer specimen, which showed several karyotypic abnormalities including an extra Y chromosome and double minutes (dmin). In early passage the karyotype of CA-HPV-10 appeared unstable with a decreasing number of cells exhibiting dmin. In late passage the dmin were replaced by a large homogeneously staining region (hsr) on 9p+ marker. The hsr was shown by FISH to be of chromosome 1 origin. The modal number was mainly hypertriploid (72, range 69 to 75). Loss of Y was remarkable (0 to 1 copy). Consistent markers included two copies each of del(1)(q12q31) and der(9)t(1;9)(?;p22), and one der(11)t(4;11) (?;q21). HPV type 18 genomic integration sites were identified on 1p for PZ-HPV-7 and on the 9p+ marker for CA-HPV-10. In conclusion, both PZ-HPV-7 and CA-HPV-10 showed clonal cytogenetic changes. These two cell lines constitute a novel in vitro model to study the mechanisms involved in human prostate carcino-genesis.

Negative control of epithelial cell proliferation by prostatic stroma.

The influence of prostatic fibroblasts on the growth of the prostatic carcinoma cell lines PC-3 and LNCaP was examined. In a double layer soft agar system, clonal growth of both cell lines was inhibited by all prostatic fibroblasts tested, irrespective of whether they were derived from malignant or non malignant prostate tissue. Irradiated fibroblasts and quiescent fibroblasts showed similar effects. The finding that growth was also inhibited in the presence of fibroblasts conditioned medium suggests that the observed effects were mediated by a diffusable growth inhibiting factor. Organ-specific production was suggested by the observation that skin fibroblasts stimulated rather than inhibited PC-3 cell growth. These findings indicate a negative control of epithelial cell proliferation by prostatic stroma.

Prostatic epithelium inhibiting factor (PEIF): organ specificity and production by prostatic fibroblasts.

The effects of prostate fibroblast conditioned medium on two prostate epithelial cell lines (PC-3, LNCaP) and on two non-prostatic cell lines (MCF-7, K562) was investigated. As prostate fibroblast conditioned medium exerts its main effect on DNA synthesis, 3H thymidine incorporation was monitored to measure factor activity. Conditioned media of all prostatic fibroblast lines investigated were inhibitory for PC-3, LNCaP and MCF-7. Conditioned medium of prostatic fibroblasts was clearly stimulatory for K562. Prostate specificity of production of PEIF was demonstrated by the fact that conditioned medium from skin fibroblasts proved to be stimulatory for PC-3. Inhibitory activity from conditioned medium as well as from a BPH homogenate was precipitated by 33-67% ammonium sulfate. These partly purified fractions were respectively five and ten times as active as "crude" conditioned medium. The physical nature of PEIF (protein or macroglycolipid) as well as the possible function (as a signal messenger between stroma and epithelium) is discussed.

Preferential loss of abnormal prostate carcinoma cells by collagenase treatment.

Effects of two different methods of tumor disaggregation on flow cytometric ploidy distribution and intact cell yield were investigated. Either mechanical disaggregation or collagenase digestion was applied to 35 prostate tumor specimens. Seven collagenase-treated samples failed to yield any intact cells, whereas with mechanical disaggregation in all cases a sufficient number of intact cells were obtained. No differences in the FCM ploidy distribution of tumors with a DNA diploid stemline were observed comparing both techniques. In DNA aneuploid tumors, however, collagenase treatment had an adverse effect on the abnormal cell populations. In 14/17 of such tumors, the abnormal cell populations were significantly reduced; in eight of these the percentage of DNA aneuploid cells declined even below the minimum percentage (10%) that was defined for DNA aneuploidy. Since collagenase is a widely used enzyme for tissue disaggregation, especially in tumor cytogenetics, the presented data will have consequences for the interpretation of results obtained by methods involving the use of this enzyme.

Inhibition of prostatic epithelial cell proliferation by a factor secreted specifically by prostatic stromal cells.

Stromal cells from the prostate were recently shown to inhibit clonal growth of the prostatic carcinoma cell lines PC-3 (hormone-independent) and LNCaP (hormone-sensitive) in coculture. Our study revealed that stromal cell-conditioned medium strongly inhibited proliferation of PC-3 and LNCaP cells when grown in monolayer culture. Antiproliferative activity was found to be reversible, and was produced specifically by prostatic stromal cells and not by stromal cells derived from skin, foreskin, uterus, kidney, and Wilms' tumor. Inhibition was not species-specific, since the cell lines AT-2.1 and MATLyLu, derived from the Dunning rat prostate tumor, were also sensitive. No inhibition, however, occurred on breast and renal carcinoma cell lines, suggesting a prostate-specific action. The putative inhibiting factor(s) could be concentrated and partially purified by ammonium sulfate precipitation. The possible role in stromal control of epithelial cell proliferation is discussed.

Characterization of chromosome 8 aberrations in the prostate cancer cell line LNCaP-FGC and sublines.

In two androgen-dependent (FGC and P70) and two androgen-independent (LNO and R) sublines of the prostate cancer model LNCaP numerical and structural aberrations of chromosome 8 were investigated in detail. The techniques used were whole chromosome paint (WCP) and fluorescence in situ hybridization (FISH) with three cosmid probes mapping to different parts of the p-arm (D8S7 (8p23.3), LPL (8p22) and PLAT (8p11.1)). By WCP all four cell lines showed four copies of chromosome 8 in most cells. However, FISH demonstrated that in all sublines deletions in the 8p region were present. The majority of both FGC and P70 had two copies of cosmids D8S7 and LPL. The cosmid PLAT showed a broader distribution (1-4 copies), especially in P70. Compared with FGC and P70, both LNO and R showed a larger number of copies (3 or 4) of all three cosmid loci. It is discussed that this difference is probably the result of nondisjunction as a reaction to loss of other sequences on 8p, possibly the tumor suppressor gene (TSG) mapping to 8p21. The fact that both sublines LNO and R are androgen-independent raises the possibility of a link between TSG loss on 8p and androgen independence.

Cytogenetic characterization of several androgen responsive and unresponsive sublines of the human prostatic carcinoma cell line LNCaP.

The cytogenetic evolution of the prostatic adenocarcinoma cell line LNCaP was investigated during long term in vitro culture. Study of five different sublines demonstrated that the original karyotype was well preserved in all sublines, with respect to the chromosome number as well as to the primary markers. All sublines showed additional, subline specific secondary marker chromosomes. Comparison of these markers in androgen responsive and nonresponsive sublines showed rearrangement of the short arm of chromosome 8 in both unresponsive sublines. The breakpoints were in 8p21 and 8p23, respectively, resulting in deletion of the 8p23----pter region in both sublines. In contrast, the hormone responsive sublines did not show any aberrations in chromosome 8. Review of published karyotypes of patients and cell lines seems to support our finding of partial deletion of 8p in androgen unresponsive prostate tumor cells.

Tissue culture loss of aneuploid cells from carcinomas of the prostate.

The frequency of aneuploid cells in cultured prostate carcinoma specimens was investigated. Ploidy distribution of the original tissue was established by flow cytometry (FCM). Fluorescence in situ hybridization (FISH) of chromosome I was applied to directly isolated and cultured cells to investigate whether any modifications in the ploidy distribution of chromosome I took place during tissue culture. In six tumor specimens that were diploid by FCM and FISH, no differences were found in the ploidy distribution of chromosome I before and after tissue culture. In eight tumors that were aneuploid by FISH, the percentage of aneuploid nuclei was significantly reduced from 28.0 +/- 15.0 (range 13-59%) in uncultured cells to 9.1 +/- 4.4 (range 4-18%) after tissue culture. The reduction of aneuploid nuclei ranged from 44 to 85%, which means that the majority of the aneuploid cell populations that were observed in the original specimens were undetectable in cultured samples. This suggests a preferential growth of normal epithelial cells. The data presented can explain the high percentage of diploid karyotypes usually found in short-term cultured prostate carcinoma specimens.

Loss and gain of chromosomes 1, 18, and Y in prostate cancer.

Nuclear suspensions of 42 prostate carcinoma specimens obtained at surgery were used to investigate loss and gain chromosomes 1, 18, and Y by fluorescence in situ hybridization (FISH) with centromere-specific probes. The outcome of FISH analysis was correlated with clinical parameters and the relationship between DNA-FCM (ploidy at cellular level) and FISH (ploidy of individual chromosomes) was assessed. Significant loss of chromosomes 1 and 18 was infrequent (respectively, three and five cases), but 53% of the tested specimens showed loss of Y. Loss was not correlated with DNA ploidy. Significant gain occurred in 36% (chromosome 1), 63% (chromosome 18), and 28% (Y) of the specimens. Gain of chromosome 18 was shown in DNA diploid (7/14) and aneuploid tumors (18/26), while gain of chromosomes 1 and Y was nearly restricted to DNA aneuploid specimens. Significant unbalance between these chromosomes occurred in 11 cases. Most cases which had significant gain of chromosome 1 or 18 showed trisomic as well as tetrasomic cells. Simultaneous loss of some and gain of other investigated chromosomes is suggestive of clonal heterogeneity and/or multiclonality. This was observed in eight tumors. Correlation between DNA-FCM and FISH was best for the Y chromosome. DNA-FCM showed more aberrant histograms with increasing stage and grade of tumors. The presence of numerical aberrations of the investigated chromosomes however, seemed independent of clinical grade or stage.

Immunocytochemical characterization of explant cultures of human prostatic stromal cells.

The study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human prostatic stromal cells and their immunocytochemical characterization. As determined by antibodies to keratin and prostate specific acid phosphatase, only small numbers (< 5%) of epithelial cells were present in primary cultures; subsequent passaging further reduced epithelial cell contamination. Antibodies against intermediate filament proteins (keratins, vimentin, and desmin) and smooth muscle actin microfilaments demonstrated that stromal cells from benign prostatic hyperplasia and prostate carcinoma differed in regard to their differentiation markers. Two contrasting phenotypes were identified in cultures derived from these two different lesions: One exhibiting fibroblastic features, was predominant in cultures derived from benign lesions and a second, showing varying degrees of smooth muscle differentiation, was more abundant in carcinoma-derived cultures. These findings are indicative of a remarkable divergence in the stromal-epithelial relationships associated with these pathological conditions and may provide us with a potential tool for studying these processes.

Identification of a homozygous deletion at 8p12-21 in a human prostate cancer xenograft.

One of the most frequent genetic abnormalities in prostate cancer is loss of the complete or part of the short arm of chromosome 8, indicating the localization of one or more tumor suppressor genes on this chromosomal arm. Using allelotyping, a frequently deleted region in prostate cancer in a genetic interval of approximately 17 cM between sequence tagged sites D8S87 and D8S133 at chromosome arm 8p12-21 was previously detected. A detailed physical map of this region is now available. Using known and novel polymorphic and nonpolymorphic sequence tagged sites in this interval, a search for homozygous deletions in DNAs from 14 prostate cancer-derived cell lines and xenografts was carried out. In DNA from xenograft PC133, the presence of a small homozygously deleted region of 730-1,320 kb was unambiguously established. At one site, the deletion disrupts the Werner syndrome gene. Data from allelotyping were confirmed and extended by fluorescence in situ hybridization analysis of PC133 chromosome spreads using centromere, YAC, and PAC chromosome 8 probes.