Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma.

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.

A 45-kDa ErbB3 secreted by prostate cancer cells promotes bone formation.

ErbB3 is a transmembrane growth factor receptor that has been implicated in the pathogenesis of human cancer. After finding that a truncated form of ErbB3 was present and upregulated in metastatic prostate cancer cells in lymph nodes and bone, we explored the pathophysiological functions of this unusual form of ErbB3 in the context of mouse calvaria as well as osteoblasts in vitro and the femur microenvironment in vivo. Here we demonstrate that prostate cancer cells expressed an alternatively spliced transcript that encodes a 45-kDa glycosylated protein (p45-sErbB3). The recombinant p45-sErbB3 purified from conditioned medium stimulated calvarial bone formation and induced osteoblast differentiation. Overexpression of p45-sErbB3 in the osteolytic prostate cancer cell line PC-3 converted its phenotype from bone lysing to bone forming upon injection into the femurs of immunodeficient mice. Further, we detected sErbB3 in plasma samples from patients with castration-resistant prostate cancer with bone metastasis. These observations establish that p45-sErbB3 is a structurally and functionally unique gene product of ErbB3 and suggest that p45-sErbB3 is likely one of the factors involved in the osteoblastic bone metastases of prostate cancer.

Low-density lipoprotein receptor-related protein 5 (LRP5) mediates the prostate cancer-induced formation of new bone.

The tendency of prostate cancer to produce osteoblastic bone metastases suggests that cancer cells and osteoblasts interact in ways that contribute to cancer progression. To identify factors that mediate these interactions, we compared gene expression patterns between two bone-derived prostate cancer cell lines that produce osteoblastic (MDA PCa 2b) or osteolytic lesions (PC-3). Both cell lines expressed Wnt ligands, including WNT7b, a canonical Wnt implicated in osteogenesis. PC-3 cells expressed 50 times more Dickkopf-1 (DKK1), an inhibitor of Wnt pathways, than did MDA PCa 2b cells. Evaluation of the functional role of these factors (in cocultures of prostate cancer cells with primary mouse osteoblasts (PMOs) or in bone organ cultures) showed that MDA PCa 2b cells activated Wnt canonical signaling in PMOs and that DKK1 blocked osteoblast proliferation and new bone formation induced by MDA PCa 2b cells. MDA PCa 2b cells did not induce bone formation in calvaria from mice lacking the Wnt co-receptor Lrp5. In human specimens, WNT7b was not expressed in normal prostate but was expressed in areas of high-grade prostate intraepithelial neoplasia, in three of nine primary prostate tumor specimens and in 16 of 38 samples of bone metastases from prostate cancer. DKK1 was not expressed in normal or cancerous tissue but was expressed in two of three specimens of osteolytic bone metastases (P=0.0119). We conclude that MDA PCa 2b induces new bone formation through Wnt canonical signaling, that LRP5 mediates this effect, and that DKK1 is involved in the balance between bone formation and resorption that determines lesion phenotype.

Antitumor efficacy of 26-fluoroepothilone B against human prostate cancer xenografts.

BACKGROUND: Epothilone compounds (e.g. epothilones A and B) represent a new structural class of microtubule inhibitors with the remarkable ability to inhibit tumor growth of multidrug-resistant cell lines at low nanomolar or even subnanomolar concentrations. Unfortunately, this therapeutic efficacy has only been achieved to date with a narrow therapeutic window. Hence, other structural analogs of compounds such as epothilone B are currently being synthesized in the hope that they will demonstrate equivalent antitumor efficacy with reduced systemic toxicity. PURPOSE: To evaluate the relative efficacy and toxicity of selectively modified epothilone compounds. METHODS: Compounds were initially screened for relative cytotoxicity against the human prostate cancer cell lines PC3, LNCaP, MDA PCa 2a and MDA PCa 2b. Growth inhibitory IC50 values of 0.5 to 4 nM were obtained. From this initial screen, one epothilone compound, 26-fluoroepothilone B, was chosen for further evaluation against the growth of s.c.-implanted MDA PCa 2b- and PC3-derived prostate tumors in athymic nude mice. The compound was administered intravenously at 2, 5 and 10 mg/kg after the tumors had reached 300 mm3. Two control groups were used: paclitaxel (40 mg/kg) and saline. RESULTS: Following treatment with 10 mg 26-fluoroepothilone B/kg, there was a sustained decrease in tumor size for 30 days reaching a maximal reduction of 80% when compared with tumor growth in the saline control group. Sustained suppression (> 20 days) of tumor growth was observed following the second drug injection. Although a maximal body weight loss of 30% occurred after the second injection, all mice completely regained their initial body weight in 20 days. A lower dose (2 mg/kg) produced a 58% maximal reduction in tumor size and a 20% body weight loss. Minimal inhibition of tumor growth, however, was obtained with paclitaxel at a maximally tolerated dose (40 mg/kg). Other epothilones tested were either less effective and/or more toxic than 26-fluoroepothilone B. This new fluorinated epothilone compound supports the growth of paclitaxel-dependent Tax-18 mutant CHO cells and produces microtubule bundles similar to those produced by paclitaxel, indicating that the two drugs share a similar mechanism of action. CONCLUSION: A new fluorinated epothilone compound, 26-fluoroepothilone B, has been described that stabilizes microtubule structures based on its support of growth of a mutant paclitaxel-dependent CHO cell line. Its antitumor activity against human prostate cancer in nude mice is superior to that of paclitaxel at equivalent toxic doses. Further research is required to determine optimal dosing strategies and to fully assess the compound's activity against other malignant diseases.

Prostate cancer cells induce osteoblast differentiation through a Cbfa1-dependent pathway.

Metastases from prostatic adenocarcinoma (prostate cancer) are characterized by their predilection for bone and typical osteoblastic features. An in vitro model of bone metastases from prostate cancer was developed using a bicompartment coculture system of mouse osteoblasts and human prostate cancer cells. In this model, the bone-derived prostate cancer cell lines MDA PCa 2a and MDA PCa 2b induced a specific and reproducible increase in osteoblast proliferation. Moreover, these cells were able to induce osteoblast differentiation, as assessed by increased alkaline phosphatase activity, Osteocalcin expression, and calcified matrix formation. This osteoblastic reaction was confirmed in vivo by intrafemoral injection of MDA PCa 2b cells into severe combined immunodeficiency disease mice. In contrast, the highly undifferentiated, bone-derived human prostate cancer cell line PC3 did not produce an osteoblastic reaction in vitro and induced osteolytic lesions in vivo. The osteoblast differentiation induced by MDA PCa 2b cells was associated with up-regulation of the osteoblast-specific transcriptor factor Cbfa1. Moreover, treatment of osteoblasts with conditioned medium obtained from MDA PCa 2b cells resulted in up-regulation of Cbfa1 and Osteocalcin expression. In support of the differentiation studies, a microarray analysis showed that primary mouse osteoblasts grown in the presence of MDA PCa 2b cells showed a shift in the pattern of gene expression with an increase in mRNA-encoding Procollagen type I and Osteopontin and a decrease in mRNA-encoding proteins associated with myoblast differentiation, namely myoglobin and myosin light-chain 2. Taken together, these findings suggest that the bone-derived prostate cancer cells MDA PCa 2a and MDA PCa 2b promote differentiation of osteoblast precursors to an osteoblastic phenotype through a Cbfa1-dependent pathway. These results also established that soluble factors produced by prostate cancer cells can induce expression of osteoblast-specific genes. This in vitro model provides a valuable system to isolate molecules secreted by prostate cancer cells that favor osteoblast differentiation. Moreover, it allows to screen for therapeutic agents blocking the osteoblast response to prostate cancer.

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms.

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.

TabBO: a model reflecting common molecular features of androgen-independent prostate cancer.

We established two human prostate cancer cell lines, MDA PCa 2a and MDA PCa 2b, the TabBO model system, that reflect common features of human androgen-independent prostate cancer that are not present in other model systems: bone origin, prostate-specific antigen production, androgen receptor expression, and androgen sensitivity. We therefore hypothesized that molecular pathways in our model system reflect common alterations responsible for the progression of a subset of human prostate cancer. Progression to androgen independence has been hypothesized to be largely associated with impairment of the regulation of cell growth or apoptosis of prostate cancer cells. Therefore, in this study, we examined molecular markers known or suspected to be important in prostate cancer progression and key regulators of cell growth and apoptosis: p53, p21WAF1/CIP1, Bcl-2, Bax, retinoblastoma (Rb), and p16INK4A/MITS1. We analyzed the expression of these markers in the cell lines, their tumor of origin, and tumors derived from the cell lines by s.c. inoculation into nude mice. DNA sequencing of the entire open reading frames of the p53 and p21 genes revealed no mutations. Additionally, accumulation of the p53 protein was not found by Western blot analysis, nor was overexpression of the Bcl-2 oncoprotein detected. Bax expression was detected in MDA PCa 2a cells, whereas it was absent in MDA PCa 2b. Rb and p16 protein expression was normal as measured by both Western blot and immunochemical analyses. Immunohistochemical studies of p53, p21, Bcl-2, and Rb in both samples from the original human cancer from which the lines were derived and mouse xenografts derived from the lines revealed similar levels of protein. These results are consistent with reports indicating that 40-50% of bone metastases of prostate cancer have wild-type p53, 50-70% do not overexpress the Bcl-2 protein, and mutations in the p21 gene are rare. Therefore, we conclude that MDA PCa 2a and MDA PCa 2b reflect molecular pathways in a common subset of human androgen-independent prostate cancer and that important molecular players in apoptosis (namely, p53 and Bcl-2) seem to be intact in this subset of androgen-independent prostate cancer. Understanding the signal-transduction pathways operating in these cell lines may help to identify therapeutic targets for prostate cancer.

Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a.

PURPOSE: We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth. MATERIALS AND METHODS: Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations. RESULTS: These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP. CONCLUSIONS: Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

p53 mutations in prostate cancer bone metastases suggest that selected p53 mutants in the primary site define foci with metastatic potential.

PURPOSE: This study was undertaken to establish the pattern of specific p53 gene mutations in prostate cancer within primary tumors and distant metastases. MATERIALS AND METHODS: We performed polymerase chain reaction-single-strand conformation polymorphism and sequencing analyses of p53 exons 5-8 in DNA extracted from 22 formalin-fixed, paraffin-embedded tissues from 17 patients. Samples from three patients included specimens from primary and metastatic sites (paired specimens). RESULTS: G:C-to-A:T transitions were the most common point mutations (64%). Six (55%) of 11 G:C-to-A:T transitions occurred at CpG dinucleotides in five hot-spot codons (175, 245, 248, 273, and 282). Sequencing analysis of the paired samples revealed that two of the three pairs had the same mutation in both. Sequencing analysis of DNA from a different area of one of the primary tumors revealed a different mutation in the p53 gene. CONCLUSIONS: Our results suggest that specific p53 mutations participate in the progression of human prostate cancer. These findings support those of others that indicate that the primary cancer is heterogeneous and clonal expansion occurs during the progression of clinically detectable prostate cancer. Our data also imply that p53 mutations at the primary site may be predictive of metastases.

Structural alterations of chromosome 5 in twelve human prostate cancer cell lines.

Neoplastic transformation, cancer progression, and metastasis are determined by a series of well-defined changes that take place in target tissue cells. Genetic alterations associated with human prostate carcinogenesis are not well defined. Some chromosomal changes, including gain of chromosomes 7, 12, 17, and X and loss of heterozygosity in chromosomes 8p, 10q, 16q, 17p, and 18q, have been reported. We examined five newly established and eight previously established prostate cancer cell lines before and after subcutis and orthotopic injection into nude mice and observed that structural alterations of chromosome 5 were present in all of the cell lines except the parental LNCaP. The fluorescence in situ hybridization preparations with the use of whole chromosome 5 DNA painting probe confirmed our Giemsa-banding data. Alterations of chromosome 5 consisted of t(1;5)(p36;q15), t(5;?)(p11;?), del(5)(q23q35) in the SP2964(= ARCaP) cell line; t(5;8)(p15;q12), i(5)(p10), t(5;15)(q11;p11) in the SP3031 cell line; t(5;?;15) (q15;?;p11), t(5;7;14)(q31;p11-q32;q11), in the SP3173 and SP3241 cell lines (derived from the same patient); del(5)(q23-33) and t(5;7;14)(q31;p11-q32;q11) in the SP3316 cell line; t(3;5)(q21;q35) in the SP2884 cell line; t(5;5)(p15;q11) in the SP2356 cell line; i(5)(p10),t(5;?)(q23;?) in the DU-145 cell line; and i(5)(p10), t(5;?)(q11;?) and t(2;5)(q15;q15) in the PC-3 cell line. Because, in most cases, alteration of chromosome 5 resulted in the partial or complete loss of 5q, we conjectured that 5q might contain one or more tumor-suppressor genes for human prostate cancer development.

Modulation of cellular proliferation and production of prostate-specific antigen and matrix adhesion molecules in human prostate carcinoma cells by polypeptide growth factors: comparative analyses of MDA PCa2a with established cell lines.

The cellular responses of a newly established and early-passage human prostate adenocarcinoma cell line, MDA PCa2a, to transforming growth factor (TGF) beta1, epidermal growth factor (EGF), and TGFalpha were characterized in terms of proliferation, production of prostate-specific antigen (PSA), fibronectin (FN) and laminin (LM). The responses of the MDA PCa2a cells were compared with those of the well-established human prostate carcinoma cell lines LNCap, PC3, and DU145. The MDA PCa2a cells were more responsive to the growth-inhibitory effect of TGFbeta1 than the established cell lines. The androgen-responsive cell lines (MDA PCa2a and LNCap) were relatively responsive to the growth-stimulatory effect of EGF and TGFalpha whereas the androgen-independent lines (PC3 and DU145) were not. Only the androgen-responsive cells produced PSA, which was further upregulated by treatment with growth factors. The androgen-independent cells did not produce PSA, and growth factors had no effect on PSA production. However, all cell lines produced abundant amounts of FN and LM, and the levels of production of these molecules were subject to modulation by growth factors. It is concluded that each growth factor elicits diverse and distinct responses in prostate carcinoma cells, which may reflect the involvement of diverse post-receptor signal pathways.

Model systems of prostate cancer: uses and limitations.

A valid experimental model system reflects the system under study and is reproducible. Model systems of prostate cancer that accurately reflect the different disease stages are necessary to ensure a proper experimental design aimed at increasing our understanding of the biology of the disease and such models are essential tools to accelerate development of new therapies for prostate cancer. Until recently, a limited number of experimental systems were available and more suitable models derived from human specimens have only recently been developed and become available for use. In addition, transgenic techniques have also permitted the development of unique mouse models. The difficulty in establishing model systems may reflect the complex requirements necessary for cancer progression and should lead us to interpret results from model systems with caution. It is unlikely that a single model system that faithfully reflects the whole process of cancer development and progression will be developed. However, thoughtful use of the available model systems will permit the study of a significant portion of prostate cancer progression. In this review we summarize the properties of the prostate cancer model systems in use and defined their utility and limitations. This review will guide the investigator seeking models with which to test specific hypotheses pertaining to prostate cancer.

Griseofulvin-induced hepatopathy due to abnormalities in heme pathway.

1. The effect of long-term griseofulvin (GRIS) topical administration on some indicators of liver damage was examined. 2. Liver porphyrin accumulation was significant; however, no porhyrin crystals were observed under light microscopy. 3. An earlier onset of hepatopathy was established (3-fold) increase of direct bilirubin values after 7 days of treatment; hepatic injury was confirmed by measuring a 6-fold increase of free bilirubin. 4. Enhanced values of alkaline phosphatase and glutamic oxalacetic transaminase (GOT) confirmed the onset of cholestasis. 5. Topical application of GRIS induced measurable hepatopathy. Nevertheless, we cannot discard the possibility that this hepatopathy could also be attributed in part to a direct reaction to xenobiotics.

Expression of bcl-2 oncoprotein and p53 protein accumulation in bone marrow metastases of androgen independent prostate cancer.

PURPOSE: We correlated the expression of bcl-2 with accumulation of p53 protein in bone marrow metastases from patients with androgen independent prostate cancer and a history of hormonal ablation therapy. These results were correlated with clinical parameters, including the extent of bone marrow metastases and patient survival. MATERIALS AND METHODS: All 43 patients studied had evidence of prostate cancer progression following androgen deprivation therapy and histologically confirmed bone marrow metastases. Decalcified tissue sections were used for immunohistochemical evaluation of bcl-2 protein and p53 protein accumulation. RESULTS: We previously established that p53 protein accumulation as detected by immunohistochemistry is a reliable indicator of p53 gene mutation in prostate cancer. Immunoreactivity was demonstrated for p53 protein in 22 of 43 cases and for bcl-2 protein in 14. A total of 28 cases (65%) exhibited immunohistochemical evidence of p53 and/or bcl-2 expression, and 15 (35%) were negative for p53 and bcl-2. The expression of bcl-2 and accumulation of p53 were independent events (p < 0.01). The expression of bcl-2 or accumulation of p53 protein in prostate cancer metastases did not significantly influence patient survival or the extent of metastatic disease. CONCLUSIONS: The presence or absence of p53 protein accumulation and/or bcl-2 expression did not correlate with tumor burden or patient survival in stage D androgen independent prostate cancer bone marrow metastases. The expression of bcl-2 protein occurs independently of and is inversely correlated with p53 mutations in advanced prostate cancer.

Establishment of two human prostate cancer cell lines derived from a single bone metastasis.

Human prostate cancer cell lines are particularly difficult to establish, and most existing cell lines do not exhibit features commonly seen in human prostate cancer. Most available models either grow only in vivo as xenografts or are androgen insensitive and fail to express prostate-specific antigen (PSA). The lack of functionally relevant model systems of advanced prostate cancer has limited prostate cancer research and therapy development. Of 30 processed samples derived from patients with prostate cancer, we established two cell lines (MDA PCa 2a and MDA PCa 2b) that express PSA and androgen receptor, grow in vitro, and are androgen sensitive. Cells from these lines produced tumors in nude mice when injected either s. c. or orthotopically (intraprostatic). Both cell lines were established from a bone metastasis of a patient whose cancer was exhibiting androgen-independent growth. Although both were derived from two samples of the same specimen, they have different genetic features (as assessed by karyotype analysis) and different phenotypes (e.g., morphology and growth rate). It is likely that they are distinct clones isolated by the use of different culture procedures and reflect the genetic heterogeneity of the tumor. These new cell lines are the first available derived from a bone metastasis of an androgen-independent prostatic adenocarcinoma that grow both in vivo and in vitro and have retained PSA expression and androgen sensitivity. They therefore constitute important model systems to address critical questions related to the androgen-independent growth of human prostate cancer and to the complex process of bone metastasis.

Zinc aminolevulinic acid dehydratase reactivation index as a tool for diagnosis of lead exposure.

The correlation between blood lead level (BLL), delta-aminolevulinate dehydratase (ALA-D) activity, and other common biochemical parameters used to assess a plumbism diagnosis have been carefully analyzed, with the aim of correctly interpreting the data handled in the laboratory. No correlation was observed between BLL and free erythrocyte porphyrins. In the case of ALA-D or Zn-reactivated ALA-D despite the direct correlation with BLL, the curve follows a potential or a logarithmic line, which is not the best to calculate BLL. The so-called Zn-ALA-D-reactivation index (iZn) has been defined as the ratio between the activity of Zn-reactivated ALA-D and the activity of ALA-D. The plot of BLL against the iZn revealed a very good linear relationship which allows an estimate of BLL with reasonable accuracy within a very wide range.

Frequency and characterization of p53 mutations in clinically localized prostate cancer.

OBJECTIVES: To determine the frequency of abnormal p53 expression and to characterize confirmed p53 mutations in tumors from patients with clinically localized adenocarcinoma of the prostate. METHODS: p53 protein nuclear accumulation was determined immunohistochemically in the initial diagnostic tumor specimens from 37 patients with clinically localized prostate carcinoma. Two primary antibodies were used on all specimens. Structural analysis of the p53 gene was performed using the methods of polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) and DNA sequencing. RESULTS: In 1 of the 37 (2.7%) tumor specimens, intense p53 nuclear staining was demonstrated using either antibody PAb 1801 or CM-1. The staining in this case was heterogeneous, with approximately 40% of tumor nuclei staining for p53. This tumor specimen was microdissected and DNA was extracted. Following PCR amplification, abnormally migrating bands were noted on SSCP analysis of exon 8. DNA sequencing confirmed the mutation as a C-->A transversion in codon 281 (asp-->glu). PCR/SSCP analysis of exons 5 through 8 was also performed for seven additional tumors that were negative for p53 nuclear accumulation by immunohistochemical (IHC) methods. All of these specimens demonstrated wild-type p53. CONCLUSIONS: The results of this study confirm and extend our previous findings that p53 mutations are rare in clinically localized adenocarcinoma of the prostate. In detecting clonal p53 mutations, standard immunohistochemical technique correlates reliably with structural p53 gene analysis of the evolutionary conserved domains encompassing exons 5-8. Importantly, most reports have demonstrated that p53 mutations detected by IHC are a late step in the progression of prostate cancer and are associated with advanced disease, dedifferentiation, and the acquisition of androgen independence.

Expression of cyclin DI in human prostate cancer cell lines.

Deregulation of cyclin expression has been found in many tumors. In this report, we studied expression of cyclin DI in three human prostate cancer cell lines: the androgen-dependent LNCaP and the androgen-independent PC3 and DU 145 cell lines. Northern blot analysis showed that DU145 and PC3 cells expressed more abundant cyclin DI than LNCaP cells. Southern blot analysis showed no evident gene amplification or rearrangement of cyclin DI in any of these cell lines. Serum starvation and replenishment were used in the cell culture to study the regulation of expression of cyclin DI. Cyclin DI mRNA expression was detected by Northern blot analysis when LNCaP cells grew in medium with serum but was not detected after serum withdrawal; however, cyclin DI mRNA was induced after serum was added. Cyclin DI mRNA expression by PC3 and DU 145 cells was detected both when they grew in medium with serum and after serum withdrawal, although expression decreased greatly after 24 hours in the PC3 cell line. Immunoprecipitation and immunohistochemical staining also showed that cyclin D I protein was always expressed in PC3 and DU 145 cells under different growth factor environment, whereas it decreased significantly in LNCaP cells deprived of serum and the level resumed again when serum was re-added. This suggests that expression of cyclin DI is regulated by exogenous growth factors in LNCaP cell line and becomes constitutive in PC3 and DU 145 cell lines.

p53 protein accumulation and gene mutation in the progression of human prostate carcinoma.

BACKGROUND: Nuclear accumulation of p53 protein has been shown to be strongly associated with missense p53 mutations. Studies of nuclear accumulation of p53 protein in prostate carcinoma cells have to date been confined to material from primary tumors. PURPOSE: We studied the accumulation of p53 protein in specimens obtained from primary and metastatic sites of prostate carcinoma. By examining the accumulation of this protein as a function of stage, histologic grade, and androgen responsiveness of the tumor, we hoped to determine the role of p53 mutation in the progression of prostate carcinoma. METHODS: The accumulation of the p53 protein in the cell nuclei was determined by immunohistochemical methods using polyclonal antibody to human p53 CM-1. The material studied consisted of formalin-fixed, paraffin-embedded tissue obtained from primary tumors and metastases of 92 patients with prostate carcinoma. Twelve samples from 11 patients were analyzed for the presence of mutations within exons 5-8 of the p53 gene (also known as TP53) by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) analysis. Sequence analysis was subsequently performed on DNA obtained by polymerase chain reaction amplification of PCR-SSCP reactions produced from six different specimens. The chi-square test, Fisher's exact test, and the Freeman Halton test were used for statistical analyses of the results. RESULTS: All tumors with p53 accumulation were metastatic (stage D), poorly differentiated, and androgen independent. Nuclear accumulation of p53 protein was strongly associated with stage (D2 versus D1 versus A-C, P < .0001), grade (Gleason score 8-10 versus 5-7, P < .003), and androgen sensitivity (androgen independent versus dependent, P < .0001). Logistic regression analysis demonstrated that androgen sensitivity predicted p53 outcome better than did stage (P < .0001) or grade alone (P < .006). There was a perfect concordance between the results obtained by PCR-SSCP analysis and the p53 protein accumulation determined by immunohistochemistry in the 12 samples studied. Mutation of the p53 gene was confirmed by sequencing DNA obtained from six specimens positive in the PCR-SSCP assay. CONCLUSIONS: p53 gene mutation is a late event in the progression of prostate cancer and is associated with advanced (metastatic) stage, loss of differentiation, and the transition from androgen-dependent to androgen-independent growth. IMPLICATION: Testing of prostate cancer biopsy specimens from metastatic sites for p53 protein accumulation and gene mutation may provide useful prognostic information and could influence the recommended course of treatment.