Prediction of metastatic potential of aspirated cells from the Dunning R-3327 prostatic adenocarcinoma model.

Pathological grading systems have failed to assess accurately the prognosis of patients with prostatic carcinoma. A visual grading system of cancer cell motility has described successfully the metastatic potential of sublines of the Dunning R-3327 rat prostatic adenocarcinoma model maintained in vitro and in vivo. Clinical application of this technique would be facilitated if it could be performed upon aspirated cells. We compared the motility of cells obtained by biopsy and fine needle aspiration from in vivo Dunning tumors and determined if the motility of aspirated cells could predict metastatic potential. Specimens from the same tumor were obtained by fine needle aspiration and incisional biopsy from three sublines of low (less than 10%) and three sublines of high (greater than 90%) metastatic potential. Membrane ruffling, pseudopodal extension and cellular translation of 100 cells were graded using time-lapse videomicroscopy. Cells obtained by biopsy and aspiration had similar average (analysis of variance, p greater than 0.7) and variation (coefficient of variation, aspirated = 18.6%, biopsy = 17.7%) of motility. Aspirated cells from three low metastatic sublines (membrane ruffling 5.20 +/- SEM 0.41, pseudopodal extension 4.10 +/- 0.57 and cellular translation 3.07 +/- 0.45) were distinguished from cells of three high metastatic sublines (membrane ruffling 7.10 +/- 0.15, pseudopodal extension 6.93 +/- 0.22 and cellular translation 5.47 +/- 0.25). Cellular translation, the best discriminator, correctly classified the metastatic potential of the subline of origin in 82% of 60 individual cells. A grading system based upon the motility of aspirated cancer cells should be studied in human prostatic carcinoma.     

Prediction of metastatic potential by a new grading system of cell motility: validation in the Dunning R-3327 prostatic adenocarcinoma model

Many grading systems for prostatic carcinoma exist; however, none allows pathologists to accurately predict the prognosis of individual patients. Examination of dead fixed histological sections of malignancies may not be the best way to predict the biological behavior of living dynamic tumors. We have developed a grading system that reproducibly characterized the motility of living cancer cells. In the Dunning R3327 rat prostatic adenocarcinoma model, three sublines of high metastatic potential (greater than 90%) were distinguished from four sublines of low metastatic potential (less than 10%). Individual cells from these sublines were correctly identified as high or low metastatic in 96% of cases by grading membrane ruffling, pseudopodal extension, and vectoral translation.     

Time lapse videomicroscopic identification of Dunning R-3327 adenocarcinoma and normal rat prostate cells

A method for accurate prediction of prognosis in human prostatic cancer does not exist. The limitations of pathologic grading systems may result from the failure of standard pathological examination of fixed dead tissue to accurately assess the biological behavior of live tumor cells. Many of the sublines of the Dunning R-3327 rat adenocarcinoma are histologically similar yet differ widely in their metastatic potential. The nonmetastatic G, occasionally metastatic AT-1 and AT-2, and highly metastatic AT-3 and MAT-Lu Dunning sublines, and normal dorsal prostate were grown in culture and filmed by time-lapse videomicroscopy. Cell membrane ruffling, undulation and pseudopodal extension, vectoral translation, irregularity of pathway, and overall subjective motility (gestalt) were visually graded. Intra-assay, intra-observer, and inter-observer reproducibility were 75, 80 and 75% respectively. The combination of ruffling, pseudopodal extension and vectoral translation was most successful in identifying the six sublines. To validate this technique prospectively, five tumor sublines and two normal prostates were graded by 10 observers unfamiliar with the technique. Fifty-nine percent of unknowns were correctly identified when motility profiles were compared to previously developed standards by least sum of squares analysis. We devised a new technique for characterizing the motility of living prostate cells which was more accurate in identifying normal rat prostate and the Dunning sublines than standard pathological examination. Prostatic cancer cell motility may reflect biological behavior and metastatic potential and thus contribute to the assessment of an individual patient's prognosis.     

Expression of a transfected v-Harvey-ras oncogene in a Dunning rat prostate adenocarcinoma and the development of high metastatic ability

To investigate the role of oncogenes in the development of metastatic ability by prostatic cancer, the viral-Harvey-ras (v-H-ras) oncogene was introduced into the Dunning rat prostate adenocarcinoma cell line, AT2.1 by means of DNA transfection. The AT2.1 cell line is a cloned cell line that is anaplastic, rapidly growing, and has low metastatic potential; after subcutaneous (s.c.) inoculation in syngeneic rats, fewer than 10% of inoculated rats develop distant metastases. Calcium phosphate mediated DNA transfections of AT2.1 cells were performed with the v-H-ras oncogene or with control DNA. The in vitro growth rate of cloned transfectants, which contain and express the v-H-ras oncogene is similar to that of untransfected AT2.1 cells and of control transfectants. After s.c. inoculation in syngeneic rats, all transfectants produced rapidly growing tumors with similar growth rates. While control transfectants had low metastatic ability comparable to untransfected AT2.1 cells, the H-ras expressing transfectants metastasized in over 80% of inoculated rats. While the mechanism by which nonmetastatic Dunning tumor sublines spontaneously develop high metastatic ability in vivo during serial s.c. passage has not been addressed in the present studies, these studies do demonstrate that expression of an activated H-ras oncogene can reproducibly convert a tumorigenic nonmetastatic prostatic cell line to a highly metastatic state.     

Expression of ras proto-oncogenes in the Dunning R3327 rat prostatic adenocarcinoma system

Steady-state levels of c-Ha-ras mRNA were measured in eight sublines of the Dunning R3327 rat prostatic adenocarcinoma. As a control, normal dorsal prostate tissue was studied. Increased expression of c-Ha-ras is associated with tumor progression in one lineage of the Dunning R3327 system (H to AT1 to MAT-Lu and MAT-Ly-Lu). Here ras mRNA increases as the tumor advances from androgen dependence and a high degree of differentiation to an anaplastic aneuploid phenotype with high metastatic potential. However, in the other Dunning lineage (H to HI to HI-F to AT3), expression of c-Ha-ras is variable and does not correlate with tumor progression. Immunocytochemistry showed that levels of the c-Ha-ras p21 protein paralleled steady-state mRNA levels in variants. Transfection assays, using NIH/3T3 cells, suggested that the ras loci were not activated in the R3327 tumors. Levels of c-Ki-ras mRNA were also measured in the Dunning tumors; these did not correlate with tumor progression in either lineage. Expression of N-ras mRNA was not detected in the Dunning tumors.     

Establishment and characterization of seven Dunning rat prostatic cancer cell lines and their use in developing methods for predicting metastatic abilities of prostatic cancers

In vitro cell lines were established from seven biologically distinct in vivo Dunning R3327 rat prostatic tumor sublines. Some of these in vitro cell lines (i.e., G, AT-1, AT-2) retain a low metastatic ability when inoculated back into syngeneic Copenhagen male rats, while others (i.e., AT-3, MAT-LyLu, MAT-Lu) retain a very high metastatic ability. A series of genetic (i.e., DNA content per cell, modal chromosomal number), as well as phenotypic parameters (i.e., morphology, 5 alpha-reductase, androgen receptor, estrogen receptor) were used to validate that the in vitro cell lines retained the major characteristics of the parental in vivo tumor sublines used for their respective establishment. A series of additional characteristics (i.e., morphology, growth rate, saturation density in surface culture, anchorage-dependent and -independent clonogenic potential) were compared between the high vs. the low metastatic in vitro cell lines to determine if a discriminatory parameter could be identified which reproducibly predicted the metastatic abilities of the particular prostatic cancer cell line. While the combination of the in vitro cell lines and their parental in vivo tumor subline will be a valuable tool for developing methods for predicting metastatic ability of prostate cancers, no single parameter yet measured is entirely successful in making this important distinction.     

Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.     

Dependence of metastatic cancer cell invasion on MLCK-catalyzed phosphorylation of myosin regulatory light chain

The role of myosin phosphorylation by myosin light chain kinase (MLCK) in regulating the invasiveness of metastatic cancer cells was investigated using the Dunning rat prostatic adenocarcinoma cell line, Mat Ly Lu, and in vitro invasion assay. Treatment with MLCK inhibitors resulted in marked reduction of invasiveness, which was principally due to impaired cellular motility, whereas the ability to survive and proliferate, to adhere to matrix, and to secrete gelatinases were minimally affected.     

Vascular endothelial growth factor content in metastasizing and nonmetastasizing Dunning prostatic adenocarcinoma

BACKGROUND: Tumor angiogenesis is important in progressive tumor growth and metastasis. In the normal rat prostate and in androgen-sensitive prostate tumors androgen ablation causes an involution of the vasculature and a decrease in the vascular endothelial growth factor (VEGF) levels before regression of the prostate gland. To examine whether angiogenesis and metastasis are regulated by VEGF in androgen-insensitive and metastasizing prostate tumors, five Dunning rat prostate cancer sublines were tested; the androgen-sensitive, nonmetastasizing R3327 PAP, and the androgen-insensitive, low metastasizing AT-1, and the three androgen-insensitive, metastasizing AT-2, AT-3, and MatLyLu Dunning prostatic adenocarcinomas. METHODS: VEGF levels were quantified in the rat dorsolateral prostate and in the five Dunning sublines using competitive RT-PCR, Western blot, and Elisa. Vascular density was determined by factor VIII staining. RESULTS: VEGF mRNA was increased in all tumors compared with normal prostates. The two metastatic sublines AT-3 and MatLyLu and the nonmetastatic subline AT-1 showed the highest VEGF mRNA expression. VEGF protein levels in the prostate gland showed increased expression in the metastatic sublines, AT-2, AT-3, and MatLyLu, compared with the nonmetastatic AT-1 subline and the ventral prostate. VEGF proteins in serum were highest in the metastatic AT-3 subline. The vessel density was highest in the two highly metastatic sublines AT-3 and MatLyLu. CONCLUSIONS: Our results suggest that VEGF levels are associated with microvessel density and the previously established metastatic pattern of these rat prostate tumor systems.     

Effect of pentosan, a novel cancer chemotherapeutic agent, on prostate cancer cell growth and motility.

Pentosan is a new chemotherapeutic drug which is currently in Phase I clinical trials. In our experimental systems, in vivo, pentosan inhibits the growth of the highly metastatic MAT-LyLu (MLL) Dunning R3327 prostate cancer cell line only at toxic doses and has no apparent effect on growth in vitro. The mechanism of tumor inhibition of this drug is unknown; however, in vitro, pentosan exhibits a potent inhibition of cell motility. Cell motility is essential for tumor cell metastasis and angiogenesis. By blocking cell motility, pentosan has the potential to inhibit both tumor growth and metastasis. We have characterized the mechanism of motility inhibition by pentosan and believe it alters cell-extracellular matrix interactions. The mechanism of motility inhibition by pentosan appears to be independent of cytoskeletal structural alterations, including changes in microfilament and microtubule networks. Pentosan acts through a different mechanism than suramin, a drug which inhibits motility through inhibition of growth factor effects. In vitro, pentosan alters cellular contacts with the extravascular matrix and inhibits cell motility. In vivo, pentosan prolongs survival of rats injected with MLL cells by 25%, but did not appear to decrease the rate of primary tumor growth or the number of metastatic lesions in the treated animals. These data suggest that, in vivo, pentosan acts through an as yet undefined mechanism.     

Genetic instability assessed by sister chromatid exchange analysis in the dunning R-3327 rat prostatic adenocarcinoma model and its relationship to metastatic potential

The original Dunning R-3327 tumor, described in 1961, has given rise to distinct sublines of different metastatic potentials. The different phenotypes cannot be explained by differences in chromosomal number, DNA content, or nuclear pleomorphism. Sister chromatid exchange is an interchange between two strands of DNA indicative of DNA damage. The frequency of sister chromatid exchanges is a well-accepted measure of genetic instability. To determine whether an assay of genetic instability could distinguish sublines capable of generating cells of the metastatic phenotype, cells from three sublines of low (<10%) metastatic potential and three sublines of high (>90%) metastatic potential were cultured in 10 μM 5-bromodeoxyuridine to label DNA. Chromosome preparations were made and sister chromatids were differentiated with Hoechst 33258 dye and Giemsa stain. Sixty metaphase spreads from each subline were scored for SCE and chromosome number. The low metastatic sublines G, AT-1, and AT-2 had 0.32 ± standard deviation 0.10, 0.38 ± 0.12, and 0.14 ± 0.05 sister chromatid exchanges per chromosome, respectively. The high metastatic sublines AT-3, MAT-Lu, and MAT-LyLu had 0.55 ± 0.17, 0.32 ± 0.1, and 0.33 ± 0.2 sister chromatid exchanges per chromosome, respectively. Subline differences in metastatic potentials cannot be explained by incidences of sister chromatid exchanges.     

Cell surface charge in predicting metastatic potential of aspirated cells from the Dunning rat prostatic adenocarcinoma model

The transplantable Dunning R-3327 rat prostatic adenocarcinoma model has provided a series of tumor variants with broad ranges of metastatic potential. We tested whether cell surface charge might be related to metastatic potential by measuring the electrophoretic mobility of live tumor cells obtained by needle aspiration. Cells were aspirated from tumors with low metastatic potential (following subcutaneous inoculation of 10(6) tumor cells the H, G and AT-1 variants had less than 5% metastases; AT-2 had 5-20%) and were compared to the electrophoretic mobility of cells aspirated from highly metastatic tumors (MAT-LyLu, MAT-Lu, AT-3 had greater than 90% metastases). Electrophoretic mobility expressed in mu/sec/volt/cm. was measured on 100 cells from each tumor subline, and the cell surface charge expressed as a zeta potential was calculated from electrophoretic mobility using the Helmholtz-Smoluchowski equation. The average zeta potential (+/- S.E.M.) for the four sublines with low metastatic potential was (-17.4 +/- 0.4 mV) compared to the three sublines with high metastatic potential (-26.5 +/- 0.7 mV), and the differences were significant (p less than .01) using the Mann-Whitney Wilcoxon test. Using a zeta potential of -20.5 mV as the cutoff between high and low metastatic potential, the sensitivity and specificity of zeta potential in predicting metastatic potential in 140 determinations on seven tumor lines were 92% and 82.5%, respectively. The predictive value of a positive test (value greater than -20.5 mV) was 80% and the predictive value of a negative test (value less than -20.5 mV) was 93%. The results support a difference in the cell surface charge between these metastatic and nonmetastatic tumors with increasing negativity at the cell surface correlating with increased metastatic potential, but not with tumor growth rates.     

Biometric assessment of prostate cancer's metastatic potential

Summary Currently, no protocol exists that can assess the metastatic potential of prostate adenocarcinoma. The reason for this is partly due to the lack of information on cellular changes that result in a tumor cell's becoming metastatic. In this investigation, attempts were made to devise a method that correlated with the metastatic potential of AT-1, Mat-Lu, and Mat-LyLu cell lines of the Dunning R-3327 rat prostatic adenocarcinoma system. To accomplish this, we applied BioQuant biometric parameters, i.e., area, shape factor, and cell motility. AT-1 had a lower shape factor and a greater area as compared with the more highly metastatic Mat-Lu subline. No significant difference in area or shape factor was detected between the AT-1 cell line and the highly metastatic Mat-LyLu line. However, the lowly metastatic AT-1 line had less motility as compared with the Mat-Lu and Mat-LyLu lines. This study revealed that metastatic potential could be partially predicted via area and shape factor and accurately predicted via cell motility.     

Immunologic aspects of the prostate

Our experiments involving the use of the Dunning R3327 adenocarcinoma as an animal model of prostatic cancer as well as clinical studies on the immunocompetence of pros-tatic cancer patients are described. Utilizing the Dunning tumor, we have demonstrated that this transplantable adenocarcinoma of the rat prostate was similar to human prostatic cancer with respect to its macroscopic and microscopic appearances, growth rate, growth differential in male and female recipients, and some of its metastatic potential. Cryosurgery was capable of destroying the primary tumor as it can in man. Both antibody and cellular immune responses could be produced against antigens associated with the tumor cells. Tumor-bearing rats treated by cryosurgery in combination with BCG were capable of producing an antitumor imunity that protected them from rechallenge. Clinical studies of prostatic cancer patients showed a diminished in vitro immunity, but the responses of the cancer patients were not significantly different from those of patients with benign prostatic disease.     

Differences in expression of oligosaccharide determinants by phenotypically distinct sublines of the Dunning 3327 rat prostate cancer

Oligosaccharides expressed by the 3327-H and 3327-MAT LyLu sublines of the Dunning rat prostate cancer model have been compared in formalin-fixed and routinely paraffin-embedded tumour tissues. Binding by lectins of defined specificity has been employed to identify expression of seven oligosaccharide structures by primary and metastatic prostatic carcinoma cells. Neuraminidase digestion was employed to reveal determinants masked by sialic acid. The presence of core Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4 determinants recognised by Con-A (Canavalia ensiformis) confirmed expression of complex-type glycoconjugates by plasma membrane and cytoplasmic components of the 3327-H tumour but only by cytoplasmic determinants within 3327 MAT LyLu variant tumour-cells. The only other oligosaccharide freely expressed by either tumour-subline was (GlcNAc beta 1----4GlcNAc beta 1----4-)n, recognised by WGA (Triticum vulgaris). Prior to neuraminidase digestion, PNA (Arachis hypogaea) (which identifies Type I oligosaccharides: Gal beta 1----3GalNAc-) bound to pseudoluminal membranes of the 3327-H tumour. However, ECG (Erythrina cristagalli) (which identifies type II oligosaccharides: Gal beta 1----4GlcNAc-) did not bind to this tumour. Unmasked Type I (Gal beta 1----3GalNAc-) and Type II (Gal beta 1----4GlcNAc-) oligosaccharides were not identified in the MAT-LyLu variant. After neuraminidase digestion, PNA-binding was identified along pseudoluminal plasma membranes within 3327-H tumours but only within the cytoplasm of 3327-MAT LyLu primary and metastatic tumour cells. Following neuraminidase digestion, ECG-binding was observed along pseudoluminal plasma membranes of 3327-H tumours and heterogeneously within the cytoplasm of primary, but not metastatic 3327-MAT LyLu tumours. Terminal alpha/beta GalNAc- residues recognised by SBA (Glycine max) were not freely expressed by either subline. These structures were readily detected along luminal membranes of 3327-H cells and weakly detected within the cytoplasm of primary but not metastatic MAT 3327-LyLu tumour cells following neuraminidase digestion. Fucosylated Type II structures Fuc alpha 1----2Gal(GalNAc)-), recognised by UEA-1 (Ulex europaeus-1) and GalNAc alpha 1----3GalNAc- structures recognised by DBF (Dolichos biflorus) were not identified as a component of either tumour subline. The different patterns of oligosaccharide expression, identified by lectin-binding, clearly differentiated between the two tumour sublines and distinguished them from normal prostatic epithelium. The Dunning 3327 rat prostatic cancer sublines offer a useful model with which to examine the relationship between cell-surface oligosaccharide structures and phenotypic variants within a defined tumour-cell population.(ABSTRACT TRUNCATED AT 400 WORDS)     

Combined therapeutic effects of conventional agents and an immunomodulator, PSK, on rat prostatic adenocarcinoma

Growth alteration effects of an immunomodulator, PSK, were investigated individually and in association with conventional chemotherapeutic agents cyclophosphamide, cisplatin and fluorouracil in an experimental prostatic cancer model. Copenhagen rats had subcutaneous tumors induced by injections of cells cultured in vitro from a highly metastatic hormonally unresponsive subline of the Dunning rat prostatic tumor, MAT-LyLu. Treatment with conventional agents and the immunomodulator agent individually and in combination began three days after tumor cell inoculation. PSK used alone was not able to significantly influence tumor growth. In appropriate doses, each conventional agent significantly retarded tumor growth. Used in combination, PSK and conventional agents retarded tumor growth locally and decreased metastatic spread of the tumor. Animals receiving combination therapy had increased life spans over those animals receiving single standard chemotherapeutic agents. Immunomodulation with PSK may enhance the antineoplastic effects of chemotherapeutic agents and offer a treatment option for hormone resistant prostatic cancer.     

Intermediate filament expression and the progression of prostatic cancer as studied in the Dunning R-3327 rat prostatic carcinoma system

To evaluate if there is any consistent relationship between the expression of intermediate filament proteins (IFP), particularly keratins, and the degree of malignancy of prostatic cancer cells, a series of nine Dunning rat prostatic cancer sublines that span the entire spectrum of progression of prostatic cancer were studied immunocytochemically by the use of a variety of antibodies specific for keratins, vimentin, or desmin. For the keratin studies, monoclonal antibodies with either a general reactivity to several keratins or highly specific for either luminal or basal epithelial cells of the normal rat prostate were used. By use of an antibody specific for luminal cell keratin 18, the luminal tumor cells of the well-differentiated, slow-growing H and HI-S sublines were positively stained. In most of the sublines with a more advanced state of progression (i.e., the moderately differentiated, moderately fast growing HI-M; the poorly differentiated, faster growing HI-F; and the anaplastic, very fast growing AT-1, AT-2, and MAT-Lu tumors), however, no expression of keratin specific for luminal cells was detected. In addition, several of the most advanced sublines (i.e., AT-1, AT-2, and MAT-Lu) were negative using any of the keratin antibodies. In contrast, several of the other sublines with the most advanced degree of progression (i.e., the anaplastic, very fast growing MAT-LyLu tumor derived from the AT-1 subline; and the anaplastic, very fast growing AT-3 tumor, derived from the HI-F subline), however, were positively stained with the keratin antibody specific for the luminal cells. By use of the keratin antibody specific for the basal cells of the normal rat prostate, the basal tumor cells of the well-differentiated slow-growing H and HI-S tumor were positively stained. This positive staining for basal cell keratin was also found in the HI-M and HI-F tumors, while the AT-1, AT-2, MAT-Lu, MAT-LyLu, and AT-3 were negative with this antibody. Thus, a loss in staining for basal cell keratin was consistently associated with the most advanced state of tumor progression. Vimentin-positive staining was demonstrated either alone or with keratin-positive staining in part of the epithelial cancer cells of all the sublines. An increase in the positive staining for vimentin was consistently associated with a more advanced state of tumor progression. Desmin-positive staining was found only in smooth cells present within the various tumor sublines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of a high-efficiency method for gene marking of Dunning prostate cancer cell lines with the enzyme β-galactosidase

Although the bacterial enzyme β-galactosidase has been used as a reporter gene in a variety of mammalian systems, the variability and instability of its expression has limited its use. Transfection of Dunning rat prostatic cell lines with β-galactosidase expression plasmids resulted in 5–10% of cells expressing the enzyme transiently, and <5% of G418-resistant clones showing any level of expression. To address this problem, we developed a labeling protocol using a replication defective retrovirus containing a β-galactosidase expression cassette. Between 30–50% of cells transduced expressed high levels of this enzyme. Homogeneous cell populations were isolated by subsequent fluorescence-activated cell sorting, using a fluorescent β-galactosidase substrate. Using a modification of standard staining procedures, small metastatic foci of cells expressing β-galactosidase in mouse lung tissue were detected with high sensitivity. This method has several advantages over standard transfection protocols, including the expedient and efficient transfer of the β-galactosidase gene and the stability of its expression in a variety of Dunning sublines. © 1996 Wiley-Liss, Inc.     

Inhibition of prostate cancer growth by estramustine and colchicine

Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of estramustine and colchicine, two microtubule inhibitors, in hormone-refractory prostate cancer. In clinically achievable concentrations, the combination of estramustine and colchicine was cytotoxic to both the Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). Microtubule function was assessed in vitro to evaluate possible mechanisms of action. In motility and cell cycle analysis assays, estramustine and colchicine inhibited cellular motility but not cell cycle transit. In vivo, these two agents both inhibited the growth of implanted Dunning rat prostate adenocarcinoma MLL cells but did not appear to have additive effects. The use of oral colchicine in the treatment of hormone-refractory prostate cancer requires further investigation.