Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)- cystine transporter: a new action for an old drug.

Although cyst(e)ine is nutritionally a non-essential amino acid, lymphoid cells cannot synthesize it, rendering their growth dependent on uptake of cyst(e)ine from their microenvironment. Accordingly, we previously suggested that the x(c)- plasma membrane cystine transporter provided a target for lymphoid cancer therapy. Its inhibition could lead to cyst(e)ine deficiency in lymphoma cells via reduction of both their cystine uptake and cysteine supply by somatic cells. In this study, using rat Nb2 lymphoma cultures, drugs were screened for growth arrest based on x(c)- inhibition. Sulfasalazine was fortuitously found to be a novel, potent inhibitor of the x(c)- transporter. It showed high rat lymphoma growth-inhibitory and lytic activity in vitro (IC50 = 0.16 mM), based specifically on inhibition of x(c)--mediated cystine uptake, in contrast to its colonic metabolites, sulfapyridine and 5-aminosalicylic acid. Sulfasalazine was even more effective against human non-Hodgkin's lymphoma (DoHH2) cultures. In rats (n = 13), sulfasalazine (i.p.) markedly inhibited growth of well-developed, rapidly growing rat Nb2 lymphoma transplants without apparent side-effects. Reduced, macrophage-mediated supply of cysteine was probably involved. In five rats, 90-100% tumor growth suppression, relative to controls, was obtained. The x(c)- cystine transporter represents a novel target for sulfasalazine-like drugs with high potential for application in therapy of lymphoblastic and other malignancies dependent on extracellular cyst(e)ine.

Intermittent androgen suppression for prostate cancer: Canadian Prospective Trial and related observations.

The Canadian Prospective Trial of intermittent androgen suppression was a prototype therapeutic initiative started in 1995 for the management of patients in biochemical relapse after radiation for localized prostate cancer. An interim analysis has yielded several observations on the relations between baseline serum prostate specific antigen (PSA), nadir serum PSA, Gleason score, and time off-treatment. In a typical androgen-dependent tumor, the response of serum PSA to androgen withdrawal is biphasic, but with early tumor progression, plateauing of serum PSA is observed. Ligand-independent activation of the androgen receptor, a mechanism subserving the initiation of androgen independence, can be counteracted experimentally with decoy molecules and clinically with nonsteroidal antiandrogens. In some patients, it is possible to lengthen the off-treatment interval by inhibiting the enzyme 5 alpha-reductase, an effect that can be reinforced by lowering serum testosterone with an antigonadotropin. Serial measurements of serum PSA indicate that intermittent androgen suppression engenders a more diverse range of hormone-related responses than previously appreciated. These include: (1) repeated differentiation of tumor with recovery of apoptotic potential; (2) inhibition of tumor growth by rapid restoration of serum testosterone; and (3) restraint of tumor growth by subnormal levels of serum testosterone. These responses are aspects of regulation that should be taken into account when planning long-term treatment of prostate cancer with intermittent androgen suppression.

Long-term neoadjuvant hormone therapy prior to radical prostatectomy: evaluation of risk for biochemical recurrence at 5-year follow-up.

OBJECTIVES: To assess the effects of 8 months of neoadjuvant therapy on pathologic stage and biochemical recurrence rates. METHODS: One hundred fifty-six men with clinically localized prostate cancer were treated with neoadjuvant combined androgen withdrawal therapy for 8 months prior to radical prostatectomy. Preoperative clinical stage, Gleason score, and serum prostate-specific antigen (PSA) levels were compared with treatment outcome (pathologic stage and PSA recurrence). RESULTS: PSA at diagnosis was 10 microg/L or higher in 36% with a mean of 11.5 microg/L. Clinical stage was T1c in 18%, T2 in 74%, and T3a in 8%. Gleason score was 6 or lower in 76% and 7 or higher in 24%. Pathologic stage was T0 in 13%, T2 in 66%, T3 (specimen confined) in 13%, T3 (margin positive) in 6%, and TxN+ in 2%. Incidence of positive margins increased with clinical stage T3a versus organ-confined disease (25% versus 4%, P <0.05), pretreatment Gleason scores 7 or higher versus Gleason scores 6 or lower (11% versus 4%, P = NS), and pretreatment PSA levels higher than 10 microg/L compared with PSA levels lower than 10 microg/L (15% versus 0%, P <0.01). Overall PSA recurrence rate was 12.2% after a mean postoperative follow-up of 54 months. Risk of PSA recurrence increased with clinical stage (25% T3 versus 11% organ confined, P <0.01), pretreatment PSA (7% if PSA lower than 10 microg/L versus 21% if 10 microg/L or higher, P <0.02), Gleason score (9% if 6 or lower versus 22% if 7 or higher, P <0.02), and pathologic stage (6% of pT2, 24% of pT3M-, and 56% of pT3M+, P <0.01). PSA recurrences occurred in 6% of patients with no adverse preoperative risk factors, 12% with any one of the high-risk factors, and 29% with any two of the high-risk factors. CONCLUSIONS: Risk of PSA recurrence after 8 months of neoadjuvant therapy is low after 5 years of follow-up and remains proportional to the presence of adverse preoperative risk factors. Prospective randomized studies are required to determine whether longer duration of neoadjuvant therapy reduces the risk of biochemical recurrence after radical prostatectomy.

Determinants of DNA sequence specificity of the androgen, progesterone, and glucocorticoid receptors: evidence for differential steroid receptor response elements.

While androgen, progesterone, and glucocorticoid receptors perform distinct physiological functions by regulating unique sets of genes, in vitro they can transactivate a common high-affinity DNA-binding target. Naturally occurring steroid response elements display nucleotide divergence that lowers binding affinity in comparison to the optimal binding element, but enhances receptor-type specificity. We investigated the role of nucleotide deviations within the DNA-binding site for contribution to steroid receptor specificity. We hypothesized that receptor specificity drives the evolution of binding site sequence, rather than strictly receptor-binding affinity. Receptor-selective targets can evolve by some nucleotides selected on the basis of additional bond energy, and others may be selected by differential tolerance to discourage binding from inappropriate receptors. To identify receptor-specific binding sites, we mimicked these dual selection pressures in a receptor-competitive environment in which DNA binding sites for the androgen or progesterone receptors were selected in the presence of the glucocorticoid receptor. These analyses also demonstrated that steroid receptors strongly select nucleotides in the spacer and flanking regions of the half-site and do so in an asymmetric fashion, indicating that steroid receptors interact with DNA in an allosteric manner that affects the transcriptional activation potential.

Selective activation of the probasin androgen-responsive region by steroid hormones.

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.

Prostate cancer: 9. Treatment of advanced disease.

A 70-year-old man is referred to a urologist for recommendations on the management of metastatic prostate cancer. His cancer was diagnosed 5 years ago, and he underwent radical prostatectomy at that time. The tumour was confined to the prostate gland (Gleason score 7), and during surgery the lymph nodes were assessed as being clear of cancer. Before the surgery, the patient's prostate-specific antigen (PSA) level had been 8 ng/mL. After the prostatectomy, PSA was at first undetectable, but recently the PSA level rose to 2 ng/mL and then, at the most recent test, to 16 ng/mL. A bone scan was ordered to investigate back discomfort, which has been persistent but easily controlled with acetaminophen. Unfortunately, the bone scan shows several sites of metastatic disease. The man's medical history includes type 2 diabetes, which has developed during the past 3 years and which is controlled by diet, as well as asymptomatic hypertension, which is managed by means of a thiazide diuretic. The patient asks what treatments are available, what impact they are likely to have on his disease and what risks are associated with the therapies.

Clinical Experience with Intermittent Androgen Suppression in Prostate Cancer: Minimum of 3 Years' Follow-Up.

This report reviews the long-term follow-up of a prospective Phase II evaluation of intermittent androgen suppression in the treatment of prostate cancer. A total of 87 patients have been entered in this protocol. At the time of this report, 50 men have been followed for a minimum of 3 years. Treatment was initiated with combined androgen blockade and continued for at least 6 months until a serum PSA nadir was observed. Medication was then withheld until the serum PSA increased to mean values between 10 and 20 ng/mL. This cycle of treatment and no treatment was repeated until the regulation of PSA became androgen independent. The total time in the study ranges from 40 to 126 months, with a mean of 65.5 months. The off-treatment period in the first cycle for the men with long-term follow-up was associated with an improvement in the sense of well-being and recovery of libido and potency in men who reported normal or near-normal sexual function before the start of therapy. The average time off therapy (percentage time off therapy) for cycles 1, 2, 3, and 4 was 15 months (54%), 10 months (48%), 8 months (45%), and 7 months (40%), respectively. The study group included 9 patients treated because of a rising PSA concentration after radiation therapy for locally advanced cancer. These patients have been off therapy for an average of 22 and 13 months in treatment cycles 1 and 2, respectively. Six patients with rising PSA values after radical prostatectomy and with follow-up exceeding 36 months have been off therapy for an average of 19 and 11 months in treatment cycles 1 and 2, respectively. Of the 87 patients, 23 have had their disease progress to androgen independence at a median of 32 months of treatment, and 13 have died cancer-specific deaths at a median of 48 months. Prostate cancer is amenable to control by intermittent androgen suppression. This approach affords an improved quality of life when the patient is off therapy, with reduced toxicity and costs. Phase II trials suggest that there is not a negative impact on patient outcome. Randomized protocols are currently in progress to determine whether survival is affected in a beneficial or adverse way by such treatment in men with locally recurrent or metastatic cancer.

Prostate cancer: molecular biology of early progression to androgen independence.

To improve the therapy for prostate cancer, it will be necessary to address the problems of progression to androgen independence and the process of metastatic spread of tumour. The complexity of the latter condition is likely to mitigate against the immediate development of relevant therapeutic approaches. However, the basis of androgen independence appears to be a problem of simpler dimensions and more amenable to treatment with current therapeutic technology. Since early tumour progression can be detected by an incomplete prostate-specific antigen (PSA) response to androgen withdrawal therapy, a study of the molecular biology of PSA gene regulation may well provide insight into new methods for preventing or delaying this problem. Mounting evidence suggests that ligand-independent activation of the androgen receptor may be one underlying mechanism of androgen independence. In the absence of androgen, a compensatory increase in the activity of cAMP-dependent protein kinase (PKA) enhances the ability of the androgen receptor to bind to the response elements regulating PSA gene expression. The activation of the androgen receptor through up-regulation of the PKA signal transduction pathway involves the amino-terminus of the androgen receptor, the function of which may be altered either by modifications such as phosphorylation, or through interactions with co-regulators or other proteins. Of therapeutic interest is the fact that this effect can be counteracted experimentally by the anti-androgen, bicalutamide, and clinically by several other similar agents. We speculate that the inhibition of PKA-activated androgen receptor might also be accomplished by decoy molecules that can bind to the relevant activated site on the amino-terminus or competitively interact with proteins recruited by the PKA pathway that are responsible for activating the receptor in the absence of androgen. Such molecules might include small mimetic substances or agents that can gain access to the nucleus of the cell.

Intermittent androgen suppression for prostate cancer: rationale and clinical experience.

The rationale behind intermittent androgen suppression (IAS) is based on: (1) observations that androgen ablation is palliative, not curative, in most patients with prostate cancer, and that quality of life must be considered; (2) the assumption that immediate androgen ablation is superior to delayed therapy in improving survival; (3) the hypothesis that if tumour cells surviving androgen withdrawal are forced into a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored and progression to androgen independence delayed. Several centres have now tested the feasibility of IAS therapy in non-randomized groups of prostate cancer patients using serum of prostate-specific antigen levels as trigger points. Clinical data suggest that prostate cancer is amenable to control by IAS and offers clinicians an opportunity to improve patients' quality of life by balancing the benefits of immediate androgen ablation (delayed progression and prolonged survival) while reducing treatment-related side effects and expense. Whether time to progression and survival is affected in a beneficial or adverse way is being studied in randomized, prospective protocols.

Differential transactivation by the androgen receptor in prostate cancer cells.

BACKGROUND: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines. METHODS: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter. RESULTS: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain. CONCLUSIONS: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.

Butyrate analogue, isobutyramide, inhibits tumor growth and time to androgen-independent progression in the human prostate LNCaP tumor model.

Progression to androgen independence remains the main obstacle to improving survival and quality of life in patients with advanced prostate cancer. Induction of differentiation may serve as a rational basis for prevention of progression to androgen independence by modulating gene expression activated by castration or upregulated during androgen-independent progression. The objectives of this study were to characterize the in vitro effects of sodium butyrate on human prostate cancer cell growth, PSA gene expression, and differentiation in the LNCaP tumor model and to determine whether tumor progression in vivo is delayed by isobutyramide, an orally bioavailable butyrate analogue with a longer half-life. The effects of isobutyramide on LNCaP tumor growth and serum PSA levels in both intact and castrate male mice were compared to controls. At concentrations >1 mM, butyrate induced dose-dependent changes towards a more differentiated phenotype, G1 cell cycle arrest, and an 80% decrease in LNCaP cell growth rates. PSA gene expression was increased threefold by butyrate, indicative of differentiation-enhanced gene expression. The half-life of isobutyramide in athymic mice was determined by gas chromatography to be 4 h. During a 4 week period in intact-placebo mice, tumor volume and serum PSA increased 4.1- and 6.6-fold, respectively, compared to twofold and 2.7-fold increases in tumor volume and serum PSA in intact-treated mice. During a 7 week period in castrate-placebo mice, tumor volume and serum PSA levels increased 2.4-fold and fourfold, respectively, compared to a 50% reduction in tumor volume and a twofold increase in serum PSA above nadir levels in castrate mice treated with adjuvant isobutyramide. Isobutyramide treatment induced pronounced morphological changes in LNCaP tumor cells, with loss of defined nucleoli and dispersion of chromatin distribution. LNCaP tumor PSA mRNA levels actually increased threefold, indicative of differentiation-enhanced gene expression. This study demonstrates that butyrate causes LNCaP cell cycle arrest and increased PSA gene expression, both indicative of differentiation. The combination of castration and adjuvant isobutyramide was synergistic in delaying tumor progression. Decreased tumor cell proliferation and increased PSA gene expression induced by isobutyramide results in disconcordant changes in serum PSA and tumor volume and reduces the utility of serum PSA as a marker of response to therapy.

Intermittent androgen suppression for prostate cancer: rationale and clinical experience.

Research on hormonal treatments of prostate cancer over the past 20 y have focused on maximizing androgen ablation through combination therapy. Maximal androgen ablation increases treatment-related side-effects and expense and fails to prolong time to androgen-independent (AI) progression. The rationale behind intermittent androgen suppression (IAS) is based on: (1) observations that androgen ablation is palliative, not curative in most patients where quality of life must be considered; (2) assumption that immediate androgen ablation is superior to delayed therapy in improving survival of patients with prostate cancer; and (3) the hypothesis that if tumor cells which survive androgen withdrawal are forced into a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored and progression to androgen independence may be delayed. Observations from animal model studies suggest that progression to androgen-independence involves adaptive responses to androgen deprivation which are, in turn, modulated by intermittent androgen replacement. Supported by these animal model observations, several centers have now tested the feasibility of IAS therapy in non-randomized groups of patients with prostate cancer using serum PSA as trigger points. Experimental and clinical data suggest that prostate cancer is amenable to control by IAS. IAS may offer clinicians an opportunity to improve quality of life in patients with prostate cancer by balancing the benefits of immediate androgen ablation (delayed progression and prolonged survival) while reducing treatment-related side effects and expense. Whether time to progression and survival is affected in a beneficial or adverse way is being studied in randomized, prospective protocols. The purpose of this article is to review the rationale behind IAS, compare observations from published series, and discuss potential indications and treatment strategies using IAS.

Increased cystine uptake capability associated with malignant progression of Nb2 lymphoma cells.

Analysis of rat, pre-T cell 'Nb2 lymphoma' sublines, manifesting different degrees of malignant progression, can indicate phenotypic changes potentially useful as therapeutic targets. In this study, the prolactin (cytokine)-dependent Nb2-11 and autonomous Nb2-SFJCD1 sublines were compared for in vitro thiol growth requirements. Whereas Nb2-11 culture growth depended on 2-mercaptoethanol (2-ME; 33-100 microM), Nb2-SFJCD1 cells were 2-ME-independent. This difference stemmed from differential uptake of exogenous L-cystine, critically required for proliferation. Uptake of 35S-L-cystine (10 microCi/ml; 40 microM) showed Nb2-11 cells had low cystine uptake capability; 2-ME enhanced cystine uptake to growth-sustaining levels. Nb2-SFJCD1 cells did not require 2-ME due to intrinsic, 11-fold higher cystine uptake via the x(c)- cystine/glutamate transport system. In absence of 2-ME, monosodium glutamate abrogated Nb2-SFJCD1 proliferation by specifically inhibiting cystine uptake (85% at 10 mM). Elevated glutathione (GSH) levels were not essential for growth of either line as shown with L-buthionine-(S,R)-sulfoximine (0.1-4 mM) treatment. The cyst(e)ine requirement therefore did not primarily involve maintenance of normal GSH levels, reported critical for T lymphocyte replication. These and other results suggest increased cystine uptake capability constitutes another potential step in progression of T cell cancers which is not coupled to cytokine autonomy or metastatic ability development. The x(c)- transport system apparently provides a novel target for T cell cancer therapy. Its inhibition would suppress cystine uptake by certain progressed cells, and also interfere with cystine uptake, and subsequent cysteine release, by eg macrophages, thought to have a role in cysteine delivery to lymphoid cells.

Androgenic induction of prostate-specific antigen gene is repressed by protein-protein interaction between the androgen receptor and AP-1/c-Jun in the human prostate cancer cell line LNCaP.

In exploring the possible mechanisms of androgen independence of prostate-specific antigen (PSA) gene expression, we investigated the effect of elevating AP-1 by both 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment and transfection of the c-Jun expression vector in LNCaP cells. Transcription of PSA is initiated when ligand-activated androgen receptor (AR) binds to a region in the PSA promoter that contains an androgen-responsive element (ARE). It was found that TPA inhibited androgen-induced PSA gene expression by a mechanism that did not alter nuclear levels of AR protein. Overexpression of AP-1 (jun and fos proteins) also inhibited androgen-induced PSA promoter activity. These observations were apparently related to the disruption of AR.ARE complexes as demonstrated by the results of electrophoretic mobility shift assays. Specifically, c-Jun inhibited the formation of AR.ARE complexes and conversely that AR-glutathione S-transferase proteins inhibited the formation of c-Jun.TPA-responsive element (TRE) complexes. Consistent with the inhibitory effect of both proteins, anti-c-Jun antibody blocked the inhibition of AR.ARE complex formation by c-Jun. A similar, but less marked, effect was obtained when anti-AR antibody was used to prevent AR inhibition of c-Jun.TRE complex formation. These findings together with results obtained from co-immunoprecipitation experiments strongly suggest that mutual repression of DNA binding activity is due to direct interaction between the two proteins and that the degree of repression may be determined by the ratio of AR to c-Jun. The mechanism of repression studied in mutant analysis experiments yielded evidence of an interaction between the DNA- and ligand-binding domains of AR and the leucine zipper region of c-Jun. Thus, the AR is similar to other nuclear receptors in its ability to interact with AP-1. This association provides a link between AP-1 and AR signal transduction pathways and may play a role in the regulation of the androgen-responsive PSA gene.

Neoadjuvant androgen withdrawal therapy decreases local recurrence rates following tumor excision in the Shionogi tumor model.

PURPOSE: The role of neoadjuvant androgen withdrawal before radical prostatectomy continues to be defined and remains investigational. It is unknown whether decreases in positive margin rates will translate into lower biochemical and local recurrence rates until long-term followup is available. We determined whether local recurrence rates after tumor excision were less with neoadjuvant compared to adjuvant androgen ablation in the Shionogi tumor model. Androgen withdrawal in this model triggers apoptosis and complete tumor regression but rapidly growing androgen independent tumors inevitably recur after 1 month. Conceptually, the ideal time for excision is after maximal castration induced tumor involution but before outgrowth of androgen resistant clones. MATERIALS AND METHODS: Shionogi tumors were allowed to grow to 1 to 2 gm. before mice were randomly assigned to group 1-tumor excision with wide margins and castration at tumor recurrence and group 2-neoadjuvant castration for 10 days followed by wide excision of the regressed tumor. RESULTS: Tumors recurred locally in 87% of group 1 mice after a median of 17 days (range 6 to 24) and all regressed completely with adjuvant castration. However, androgen independent tumors recurred in 92% of mice after a median of 36 days. In group 2 androgen independent disease recurred in 44% of mice after a median of 40 days. Tumor-free survival was significantly greater in group 2 than in group 1 (56 versus 20%, respectively, p <0.05). Inked surgical resection margins were positive in two-thirds of group 1 and one-third of group 2 mice. CONCLUSIONS: Neoadjuvant androgen withdrawal decreases local recurrence and positive margin rates by 50% after tumor excision in the Shionogi tumor model. Although this model cannot address the issue of effect of neoadjuvant therapy on subclinical metastases, extrapolation of these results to the clinical disease suggests that neoadjuvant therapy may help to decrease not only the positive margin rates but also the subsequent risk of local recurrence following radical prostatectomy.

A metastatic and androgen-sensitive human prostate cancer model using intraprostatic inoculation of LNCaP cells in SCID mice.

Several metastasizing murine and human animal models for prostate cancer are available. However, these models are androgen-independent and lack differentiated features such as androgen receptor and androgen-regulated gene expression like prostate-specific antigen (PSA). The objective of this study was to develop a metastasizing prostate cancer model with differentiated features using the human LNCaP cell line. Athymic and SCID mice were injected either s.c. or intraprostatically with 1 x 10(6) LNCaP cells. Changes in serum and tumor PSA mRNA levels were determined before and after castration to assess time to androgen-independent progression. Local tumor and metastatic growth was assessed at sacrifice after 12 weeks. Reverse transcription-PCR (RT-PCR) was used to detect circulating LNCaP cells. LNCaP tumor incidence after s.c. injection was 100% (65 of 65) in SCID mice and 80% in athymic mice. No lymph node or distant metastases were observed with s.c. tumors, and RT-PCR for PSA transcripts was negative. Primary tumor incidence after intraprostatic injection was 89% (39 of 44) in SCID mice and 60% in athymic mice. In 10 SCID mice with primary tumors followed for 12 weeks, retroperitoneal or mediastinal lymph node metastases were found in 100%, and microscopic pulmonary metastases were identified in 40%. RT-PCR for PSA transcripts was positive in 3 of 10 mice tested. Serum PSA levels in mice with s.c. and intraprostatic tumors decreased by 65% to nadir levels at 7 and 4 days after castration, respectively. Serum PSA and LNCaP tumor PSA mRNA levels increased to precastration levels earlier in SCID mice with intraprostatic tumors compared to those with s.c. tumors. Intraprostatic injection of LNCaP cells in SCID mice provides a useful animal model to investigate mechanisms of metastasis and to evaluate therapies targeted toward inhibiting the metastatic cascade.