Isolation and characterization of transforming growth factor beta response variants from human prostatic tumor cell lines.

In this study we examined the relation between the response to transforming growth factor beta (TGF beta 1) in vitro and the growth in vivo of 1-LN-PC3-1A (1-LN) human prostatic carcinoma cells. 1-LN cells resistant to the growth-inhibitory effects of TGF beta 1 were isolated after exposure to 2 ng/ml TGF beta 1 in an anchorage-independent growth assay. Cloning of TGF beta 1-resistant and -sensitive populations produced 2 clones (R2-6 and 1-LN clone 4), which maintained relatively stable resistance or sensitivity, respectively, in the absence of TGF beta 1 for up to 12 passages. Colony formation by the R2-6 cells in the presence of TGF beta 1 was 2-10 times greater than that of 1-LN clone 4, depending upon the TGF beta 1 concentration. Injection of 1 x 10(5) R2-6 cells into athymic nude mice produced tumors with a significantly shorter latency interval as compared with 1-LN clone 4 tumors (P < 0.0001). Western immunoblotting showed that higher levels of latent TGF beta 1 protein were secreted into the culture medium by 1-LN clone 4 cells. Acidified conditioned media from both clones inhibited mink lung epithelial cell DNA synthesis. Neutralizing monoclonal antibody to TGF beta 1 but not TGF beta 2 abrogated this inhibitory effect. Comparison of the different sensitive and resistant clones showed that in vitro sensitivity to TGF beta 1 and in vivo tumor latency interval were not invariably correlated. Thus, the TGF beta 1 response phenotype in vitro was not always predictive of growth delay in vivo.     

Estrogenic action of commonly used fragrant agent citral induces prostatic hyperplasia

Summary A rat model for benign prostatic hyperplasia in man (BPH) was investigated. Citral treatment of male Copenhagen rats for 4 months via the transdermal route resulted in a marked hyperplasia of glandular epithelium and interglandular stroma in the ventral prostate. Despite the cellular hyperplasia there was not a significant increase in prostate weight. Investigations of the mechanism of action of citral showed that application of citral directly to the vagina in female, ovariectomized rats resulted in an increased proliferation of vaginal epithelium and a significant increase in the BrdUrd incorporation in vaginal epithelial cells, in short a similar effect to that of estrogen application. In an in vitro assay citral proved to inhibit estrogen binding to estrogen receptors, while no such inhibition was observed with testosterone for androgen receptors. These observations together with the estrogen implication in the BPH and the reported incidence of gynccomastia following exposure to geraniol, a precursor of citral, strongly suggest that the prostatic hyperplasia-inducing capacity of citral may be due to its estrogenic action.     

Autoradiographic and cytochemical localization of androgen in human prostatic cancer cell lines

For basic studies of receptor dynamics in androgen-responsive tissues and cells, the autoradiographic and cytochemical procedures were applied to cultured tumor cells (DU-145 and PC-3). Uptake and retention of 3H-R1881, a potent synthetic androgen, were observed in DU-145 cells. The radioactive labelling was intense, and solely confined to the nuclei of DU-145 cells. Radioactivity over PC-3 cells was minimal. For assessing binding specificity, DU-145 cells were incubated with 3H-R1881 in the presence or absence of either unlabelled R1881, testosterone, progesterone, estradiol-17 beta, or corticosterone. The displacement of 3H-R1881 with R1881 and testosterone was significant, while no displacement was observed with other steroids. Nuclear localization of cytochemical staining of the dihydrotestosterone-peroxidase conjugate was evident in DU-145 cells. Our results indicate that androgen receptor may reside primarily in target cell nuclei of androgen-responsive tissues and tumors.     

Establishment and characterization of seven human renal cell carcinoma cell lines

OBJECTIVE: To establish human renal cell carcinoma (RCC) cell lines, and to investigate the cell phenotypes and molecular characteristics of human RCC cell lines and their corresponding tumour tissues. MATERIALS AND METHODS: Seven human RCC cell lines from pathologically proven RCCs were established. The histopathology of the primary tumours, in vitro growth characteristics and status of tumour suppressor genes, mismatch repair genes and microsatellite instability (MSI) were examined in cell lines and their corresponding tumour tissues. Five of the cell lines were derived from clear cells (SNU-228, -267, -328, -349, and -1272), one from granular cells (SNU-482), and one from mixed clear and granular cell types (SNU-333). The mutational status was compared for von Hippel-Lindau (VHL), p53, TGF-beta type II receptor (TGF-betaRII), hMSH2, and hMLH1 genes in the cell lines and their corresponding tumour tissues. The MSI status of the cell lines was determined by screening for adenine repeat sequences, e.g. BAT-25, BAT-26, and BAT-40. RESULTS: All lines showed different doubling times and were confirmed by DNA fingerprinting analysis to be unique. Contamination by mycoplasma or bacteria was excluded. In two cell lines (SNU-349 and -1272) and their tumour tissues, mutations in the VHL gene were found. The SNU-267 line had a frameshift mutation in the p53 gene. A missense mutation of the TGF-betaRII gene was detected in the SNU-1272 line and the corresponding tissue. Analysis of the repeat sequences showed one cell line (SNU-349) to have MSI and the other six to have microsatellite stability. As MSI is a hallmark of the inactivation of mismatch repair genes, the presence of hMSH2 and hMLH1 mutations was investigated in all seven cell lines. An inactivating homozygous single base-pair deletion of the hMLH1 gene was found only in the SNU-349 cell line and corresponding tissue. Moreover, a frameshift mutation within an 8-bp polyadenine repeat present in the hMSH3 coding region was found only in the MSI cell line and tumour tissue. CONCLUSION: These newly established RCC cell lines should provide a useful in vitro model for studies related to human RCC. The SNU-349 cell line should be especially useful for studies of MSI and mismatch repair-defective RCCs.     

Molecular characterization of human prostate carcinoma cell lines

BACKGROUND: This study presents a comprehensive survey and characterization of available prostate carcinoma cell lines, most of which have been widely used but are incompletely characterized. METHODS: A total of 21 cell lines were investigated, including three "classical" (DU 145, LNCaP, and PC-3) and 18 "non-classical" lines (1013L, 22Rv1, ALVA-55, ALVA-101, ARCaP, CWR-R1, DuCaP, DuPro-1, LAPC-4, MDA PCa 1, MDA PCa 2a, MDA PCa 2b, NCI-H660, PC-346C, PC-93, PSK-1, UM-SCP-1, and VCaP). Cytogenetics, DNA profiling, expression of basal, luminal, and neuroendocrine differentiation markers, and mutation analyses of the TP53 and androgen receptor (AR) genes were performed. RESULTS: Based on cytogenetics and DNA profiling analyses, out of the 18 "non-classical" lines, six were confirmed to be unique, eight (in four pairs) were confirmed to be related in origin, and four lines were identified as cross-contaminants. Of this latter group, PC-93 was found to be a derivative of HeLa, whereas DuPro-1, ALVA-55, and ALVA-101 were derivatives of PC-3. The 17 genuine prostate cell lines expressed keratin 8 (K8) and K18. Nine showed AR expression, of which five harbored mutations in the AR gene. Prostate-specific antigen and DD3 were exclusively detected in AR expressing cell lines. Seven lines expressed the basal cell marker K5, three of these lines showed co-expression of AR. CONCLUSIONS: This study defines a collection of 17 genuine prostate carcinoma cell lines. This collection, although small, constitutes a variety of different types and stages of prostate cancer, while it also partly reflects the heterogeneous nature of this malignancy.     

Immortalized and tumorigenic adult human prostatic epithelial cell lines: Characteristics and applications part 2. Tumorigenic cell lines

This is Part 2 of a three-part review and deals with tumorigenic cell lines. Several immortalized and malignant adult human prostatic epithelial cell lines have been recently developed. The three most widely used carcinoma cell lines—DU-145, PC-3, and LNCaP—developed between 1977 and 1980, have greatly contributed to our current understanding of prostate cancer. Before a cell line can be accepted as having prostatic epithelial origin, some basic characteristics must be established. Expression of specific cytokeratins but absence of desmin and factor VIII should be first determined to establish epithelial origin. Responsiveness to androgens and expression of androgen receptor and prostate-specific antigen should be examined under stringent culture conditions to establish prostatic epithelial origin. Response to growth factors and expression of their receptors facilitates further characterization of cell behavior. Cell lines immortalized by human papillomaviruses (HPVs) are of special interest because HPVs are involved in a variety of anogenital cancers and may also play a role in prostate carcinogenesis. Malignant transformation of HPV-18 immortalized cells with the ras oncogene provides cell systems for investigating the multistep process of carcinogenesis. Each cell line has some unique characteristics, whether it arose directly from a carcinoma or resulted from immortalization with Simian virus 40 (SV40) or HPV, or was transformed in vitro by oncogenes. Comparisons of these characteristics should facilitate elucidation of the mechanisms involved in the initiation, promotion, and progression of prostate cancer. These cell lines will further serve as useful models for investigating tumor progression, invasion, metastasis, new therapeutic strategies, drug resistance, and its reversal and chemoprevention. The nontumorigenic cell lines were discussed in Part 1 [1]. This review summarizes the characteristics of several currently available tumorigenic, adult human prostatic epithelial cell lines. Prostate 30:58–64, 1997 © 1997 Wiley-Liss, Inc.     

Local hyperthermia for prostatic disease: in vitro studies on human prostatic cancer cell lines.

Local hyperthermia for benign and malignant prostatic disease remains largely empirical. In an attempt to understand the biological action of hyperthermia, and its potentiation by antiandrogen seen in clinical practice, the interaction of the two has been studied in prostatic cancer cell lines. Human prostatic cancer cell lines LNCaP and DU 145 were studied to examine the effects of heat shock treatment (HST), androgen (5 alpha-dihydrotestosterone: 5 alpha DHT) and antiandrogen (hydroxyflutamide: OH-Flut) on cell growth and survival. Response (measured as increased DNA content) to 5 alpha DHT demonstrated that LNCaP was androgen sensitive, whereas DU 145 was androgen insensitive; OH-Flut stimulated LNCaP growth but had no effect on DU 145 growth. Thermotolerance was exhibited by DU 145 cells but not by LNCaP cells. The combination of HST followed by OH-Flut markedly reduced survival of LNCaP cells compared with HST alone. This effect was not observed in DU 145 cells. The enhanced cytotoxic effect of antiandrogen and hyperthermia could minimise the effect of thermotolerance in malignant cells surviving initial hyperthermia treatment and might suggest real clinical value for the combination or sequence.     

Phenotypic characterization of immortalized normal and primary tumor-derived human prostate epithelial cell cultures

BACKGROUND: Cell lines can provide powerful model systems for the study of human tumorigenesis. However, the human prostate cancer cell lines studied most intensively by investigators (PC3, DU145, and LNCaP) were established from metastatic lesions, and it is unlikely that they accurately recapitulate the genetic composition or biological behavior of primary prostate tumors. Cell lines more appropriate for the study of human prostate primary tumors would be those derived from spontaneously immortalized cells; unfortunately, explanted prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we examined whether cell lines developed through viral gene-mediated immortalization of human normal or primary tumor prostate epithelium express aspects of the normal or malignant phenotypes, and could serve as appropriate models for normal or transformed human prostatic epithelium. METHODS: To accomplish these goals, we assessed the phenotypic expression of cell cultures established through the immortalization of normal (1532N, 1535N, 1542N, and PrEC-T) or malignant (1532T, 1535T, and 1542T) human prostate epithelium with the E6 and E7 genes of HPV-16, or the large T antigen gene of SV40. RESULTS: Examination of these cell lines for their proliferative rates and their abilities to grow with or without serum or androgen stimulation, to form colonies in soft agar, or to form tumors in vivo, suggests that they may serve as valid, useful tools for the elucidation of prostate tumorigenesis. Moreover, the observation of structural alterations involving chromosome 8, including gain of 8q in 3 of the 4 cell lines expressing aspects of the malignant phenotype, implies that these cell lines accurately recapitulate the genetic composition of primary prostate tumors. CONCLUSIONS: Taken together, these data suggest that cell lines generated from immortalized normal or primary tumor epithelium may be useful for the elucidation of early transforming events in the prostate.     

Establishment and characterization of cell lines derived from a human osteosarcoma

Continuously growing cell lines have been established in vitro from a human osteosarcoma after transplantation into athymic nude mice. These cell lines grew as an adherent monolayer and consisted of various types of cells. Twelve clones were colonially isolated from a cell line, HuO-3N1, and subdivided into three groups depending on the morphologic features. The cells of HuO-3N1 and its clones had alkaline phosphatase (ALP)-positive granules in the cytoplasm. Cytogenetic studies showed that these cell lines were human aneuploid lines. A tumor was produced by injection of HuO-3N1 cells into an athymic nude mouse. ALP activity increased in a clonal cell line, HuO-3N1 cl-2, when cells were treated with 1,25-dihydroxy-vitamin D3. The proliferation of cells was inhibited when the cells were cultured in a medium supplemented with L-homoarginine, which is an inhibitor of bone and liver-specific ALP. This cell line has an osteoblastic phenotype and provides a useful model for studies of human osteosarcoma and phenotypical expression of human osteoblastic cells.     

Epidermal growth factor receptor mRNA levels in human prostatic tumors and cell lines.

The mitogenic activity of epidermal growth factor (EGF) is mediated by a cell surface receptor (EGF-R) which has been identified in human prostate tissues. Because of conflicting reports on the relative levels of EGF-R in prostate tumors as measured by binding of radiolabelled EGF, we have examined EGF-R expression at the level of the specific messenger RNA using a sensitive RNase protection assay. Expression of the mRNA for EGF-R was higher in carcinoma (CaP, N = 38) than in benign prostatic hyperplasia (BPH, N = 35) samples (p less than 0.01). The highest levels of EGF-R mRNA were found in the human prostatic carcinoma cell lines, PC-3 and DU145. Among the CaP samples, there was an association of higher EGF-R mRNA levels with higher tumor extent and dedifferentiation. Since EGF has also been found in prostatic tissues, the enhanced expression of the EGF-R gene may play a role in the growth of prostate tumors, possibly by an autocrine pathway.     

Effects of bombesin and its antibody on growth of human prostatic carcinoma cell lines]

Bombesin (BMBS), a tetradecapeptide isolated from frog skin, and its antibody were evaluated in vitro and in vivo for their growth modulating effects on human prostatic carcinoma cell lines DU-145 and PC-3. The two cell lines were maintained in DME medium containing 2% FCS and 1 microgram/ml insulin in a humidified atmosphere of 5% CO2/95% air at 37 degrees C. BMBS added at 0.1-10.0 nM caused a striking shift of the concentration-dependent increase in cell growth of DU-145 and PC-3 in the absence of any other exogenously added growth factors. The addition of 1:250 antibody versus BMBS (Ab-BMBS; rabbit antiserum) to the medium during the lag phase of growth resulted in specific inhibition of growth of DU-145 and PC-3 with or without BMBS. Nude mice were transplanted with DU-145 cells in order to evaluate the suppressing qualities of Ab-BMBS on carcinoma cells in vivo. After the inoculation of 10(7) cells/mouse DU-145 into nude mice, 20 microliters/mouse Ab-BMBS was intraperitoneally injected into mice three times weekly for three weeks. In mice administrated with Ab-BMBS, the growth of DU-145 was suppressed markedly. By immunocytochemical study, BMBS immunoreactivities were detected on DU-145 and PC-3 cells. These results suggest that BMBS can function as an autocrine growth factor for human prostatic carcinoma cells. Furthermore, on xenografts in mice, Ab-BMBS inhibits the increase of human prostatic carcinoma.     

Immortalized and tumorigenic adult human prostatic epithelial cell lines: Characteristics and applications part I. Cell markers and immortalized nontumorigenic cell lines

Several immortalized and malignant adult human prostatic epithelial cell lines have recently been developed. The three most widely used carcinoma cell lines, DU-145, PC-3, and LNCaP, developed between 1977 and 1980, have greatly contributed to our present understanding of prostate cancer. Before a cell line can be accepted as having prostatic epithelial origin, some basic characteristics must be established. Expression of specific cytokeratins, but absence of desmin and factor VIII, should be first determined to establish epithelial origin. Responsiveness to androgens and expression of androgen receptor and prostate specific antigen should be examined under stringent culture conditions to establish prostatic epithelial origin. Response to growth factors and expression of their receptors facilitates further characterization of cell behavior. Cell lines immortalized by human papillomaviruses (HPVs) are of special interest because HPVs are involved in a variety of anogenital cancers and may also play a role in prostate carcinogenesis. Malignant transformation of HPV-18 immortalized cells with the ras oncogene provides cell systems for investigating the multistep process of carcinogenesis. Each cell line has some unique characteristics, whether it arose directly from a carcinoma or resulted from immortalization with simian virus 40 (SV40) or HPV or was transformed in vitro by oncogenes. Comparisons of these characteristics should facilitate elucidation of the mechanisms involved in initiation, promotion, and progression of prostate cancer. These cell lines will further serve as useful models for investigating tumor progression, invasion, metastasis, new therapeutic strategies, drug resistance, and its reversal and chemoprevention. This review will be published in three parts and will summarize cell markers necessary for characterization, as well as the characteristics and some applications of the immortalized as well as malignant adult human prostatic epithelial cell lines. Part 1 deals with cell markers and the immortalized, nontumorigenic cell lines. © 1996 Wiley-Liss, Inc.     

The effect of hyperthermia on human prostatic carcinoma cell lines: evaluation in vitro

The effect of hyperthermia on established human prostate carcinoma cell lines (PC-3, DU-145) and related sublines (1-LN, 125-1L) was investigated in vitro. Cells were exposed to heat treatment at 43C or 37C for varying time intervals, (one hr or two hrs) and cell survival was evaluated by the colony formation assay and by measurement of cellular growth rate. While one hr exposure at 43C did show a mean inhibition of colony formation, ranging from 29 to 41%, a statistically significant increase in inhibition rate (p less than 0.001) was observed at two hr exposure, ranging from 57 to 92%. This study is a report of the cytotoxic effect of hyperthermia on established human prostatic tumor cell lines. These in vitro results indicate that hyperthermia may become a potentially useful form of adjunctive therapy for local control of prostatic cancer. However, the temperature and exposure time may have an important impact on cell kill when this new modality for cancer treatment is proposed for a clinical trial.     

Effect of radiation combined with hyperthermia on human prostatic carcinoma cell lines in culture

The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses followed by heat treatment at 43 degrees C for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation and heat produced greater cytotoxicity than predicted from the additive effects of the two individual treatment modalities alone. These results indicate that a combined treatment regimen of radiation plus hyperthermia (43 degrees, 1 hr) might be an important tool in maintaining a better local control of prostatic cancer.     

Estramustine-induced mitotic arrest in two human prostatic carcinoma cell lines DU 145 and PC-3

In growth proliferation experiments on two human prostatic carcinoma cell lines, DU 145 cells were found to be more sensitive to the cytotoxic effect of estramustine and nor-nitrogen mustard than PC-3 cells. Estramustine was, however, much more cytotoxic in both cell lines than nor-nitrogen mustard. Cytogenetic experiments revealed that estramustine produced a drastic increase of the mitotic index in both these cell lines. This increase could be accounted for by the arrest of cells in their first treatment-metaphase. The arrested metaphases exhibited all the characteristics commonly found for stathmokinetic agents such as colchicine and vinca-analogues. No mitotic arrest was found for nor-nitrogen mustard but chromosomal aberrations were found at toxic concentrations. Estradiol exhibited minimal toxicity and caused no mitotic arrest in these cell lines. The mitotic arrest induced by estramustine was found to be reversible on removal of the drug.