High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

Increased expression of the interleukin 2 (IL-2) receptor beta chain (p70) on CD56+ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL-2 binding.

The expression of the 70-kD beta subunit of the interleukin 2 receptor (IL-2R) has been examined on peripheral blood lymphocytes (PBL) obtained from patients receiving systemic infusions of IL-2. Using monoclonal antibodies directed against p70, flow cytometric analyses revealed a greater than threefold increase in expression of the IL-2R beta chain on CD56+ natural killer (NK) cells from post-IL-2 therapy PBL relative to pre-therapy cells. The level of p70 expression on the post-therapy cells was three- to fourfold greater (based on fluorescence intensity) than the level of p70 expression on YT cells, an NK-like cell line that expresses approximately 12,000 intermediate affinity IL-2 binding sites/cell. Despite the high level of p70 expression, in 125I-IL-2 binding assays only 790-1,290 intermediate affinity IL-2 binding sites/cell were detected on post-therapy cells from six patients. These data represent the first report of increased p70 expression after in vivo IL-2 administration and suggest a requirement for at least one additional subunit for the formation of functional intermediate affinity IL-2Rs. Furthermore, the presence on the surface of post-therapy NK cells of excess p70 that does not bind IL-2 with intermediate affinity implies that the formation of intermediate affinity IL-2Rs is not solely determined by the level of p70 expression, and that the response of NK cells to IL-2 might be regulated by altering the expression of p70 or some other IL-2R subunit.

Determination of thermal histories of archeological cereal grains with electron spin resonance spectroscopy.

The thermal histories of archeological cereal grains were examined by electron spin resonance spectroscopy. Studies with modern samples of heated cereal grain showed that the parameters of the electron spin resonance signal characterize the maximum temperature to which the sample had previously been heated. This technique has applicability in archeology and other disciplines.

Compositional mapping of cholesteryl ester droplets in the fatty streaks of human aorta.

Frozen sections prepared from human aortic tissue containing fatty streak lesions were examined on a thermally controlled stage with a polarizing light microscope. Distinct birefringent droplets, 0.5-5 mum in diameter, were observed, many apparently aggregated into clusters. The clusters were about 20 X 20 mum in diameter (the approximate size of foam cells). Upon being heated, each smectic droplet exhibited a sudden change of birefringence, indicating a change of state. The transition temperatures were compared to assess compositional distributions in the tissue. We found that for 52% of the clusters the standard deviation of the cluster's droplet melting point distribution was less than half that observed in the surrounding microscopic field. If clusters were intracellular lipid inclusions, this observation indicates that the lipid composition within a foam cell is more homogeneous than that of the overall field. However, using statistical methods, we compared droplet melting populations from cluster to cluster and found significant heterogeneity. The observations can be interpreted to suggest that many foam cells modify the cholesteryl ester fatty acid composition of their accumulations be selective uptake, temporal sampling, or chemical reaction. Furthermore, the intercellular heterogeneity suggests that different cells in the lesion may have different metabolic and transport enzyme affinities or be in different states.

Molecular organization of the cholesteryl ester droplets in the fatty streaks of human aorta.

X-ray diffraction patterns from human arterial specimens containing atherosclerotic fatty streak lesions exhibited a single sharp reflection, corresponding to a structural spacing of about 35 A. Specimens without lesions did not. When specimens with fatty streaks were heated, an order-to-disorder phase transition was revealed by the disappearance of the sharp reflection. The transition was thermally reversible and its temperature varied from aorta to aorta over a range from 28 degrees to 42 degrees C. Since cholesteryl ester droplets are a major component of fatty streaks, comparison studies were made of the diffraction behavior from pure cholesteryl esters. We found that the diffraction patterns of the fatty streak material could be accounted for by the organization of the cholesteryl esters into a liquid-crystalline smectic phase that melts from the smectic to a less ordered phase upon heating. When combined with the conclusions of others from polarized light microscopy, our study shows that a droplet in the smectic phase has well-defined concentric layers of lipid molecules. In each layer, the long axes of the molecules have a net radial orientation with respect to the droplet, but the side-to-side organization is disordered. We suggest that the accessibility of portions of the lipids for specific binding to enzymes or transport proteins may be restricted when they are in the smectic state, and that exchange of lipids with surrounding membranes or other potential binding sites may likewise be inhibited. The restriction in the smectic phase should be greater than in the less ordered phases that exist at higher temperatures.

Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4.

Previously, Puri et al. (Puri, R. K., M. Ogata, P. Leland, G. M. Feldman, D. Fitzgerald, and I. Pastan. 1991. Cancer Res. 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin. In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4. By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM. Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb. IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line. The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines. Tumor cell growth, as measured by [3H]thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner. A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4. Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro. In fact, IL-4 caused modest stimulation of their growth. Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.

Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction.

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.

Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth.

Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase (12-LOX) and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-LOX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(S)-hydroxyeicosatetraenoic acid metabolite. In vitro, 12-LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migration and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mechanism.

Cytotoxic effect of anti-Mr 67,000 protein immunotoxins on human tumors in a nude mouse model.

We have studied the potential use of immunotoxins (ITs) for therapeutic treatment of human tumors in an experimental model of human neoplasia. We tested intact ricin IT for its antitumor activity against established tumors. CEM, a human T-cell leukemia line expressing an Mr 67,000 cell surface antigen, and Daudi, a human B-cell lymphoma line which does not express the antigen, were found to be consistently tumorigenic in nude mice. ITs were synthesized using T101, a high-affinity monoclonal antibody reacting with the Mr 67,000 protein determinant and intact ricin. We have shown for the first time that established CEM solid tumors in nude mice will regress following intratumoral injection of T101-ricin IT, while Daudi tumors will not. Selective activity of T101-ricin is dependent on systemic i.v. administration of lactose and local intratumoral injection of the T101-ricin IT with lactose. Intact ricin ITs require the presence of lactose to block native ricin binding and render them antigen specific when linked to monoclonal antibody. Killing of target was cell specific since (a) nonspecific (irrelevant) ITs did not cause the regression of CEM tumors, and (b) injection of large amounts of free T101 antibody prior to T101-ricin IT blocked antitumor activity. Selectivity was not absolute, since regression occurred in one of six animals given irrelevant IT, and blocking was observed in two of four mice. Intratumoral IT treatment with 1 or 2 micrograms of T101-ricin IT plus lactose was not harmful to mice in contrast to intratumoral ricin treatment, which killed all treated tumor-bearing mice at a dose of 0.3 micrograms. Without i.v. injection of lactose, intratumoral injection of T101-ricin IT was also effective in eliminating established tumors. However, this treatment did not result in the selective elimination of tumor, since Daudi tumors also regressed following T101-ricin IT treatment. IT, made with ricin A chain only (T101-A chain IT), was also tested against established CEM tumors. We found that high dosages of T101-A chain IT did not destroy CEM tumors when injected intratumorally, even in the presence of activating agents such as NH4Cl or the carboxylic ionophore X-537 A. In contrast, in vitro experiments demonstrated that T101-A chain IT plus activating agents had potent and selective cytotoxic effect against CEM cells. We conclude that ITs are specifically toxic to established tumors. Although selectivity is not absolute, ITs exhibit potential as a new class of antitumor reagents.

Combined immunochemotherapy of human solid tumors in nude mice.

In vivo immunochemotherapy of human solid tumors was studied in a nude mouse model. Large tumors (3 to 6 cm3) were induced by s.c. injection of the acute lymphoblastic leukemia T-cell line CEM. Transient tumor inhibition could be achieved by intratumoral injection of an intact-ricin immunotoxin that specifically recognizes a determinant CD5 (T,p67) expressed on the cell surface. Injection of the in vitro active cyclophosphamide congener mafosfamid had little effect on the progression of tumor growth. A combination regimen of immunotoxin and mafosfamid induced the most dramatic antitumor effect; a 72 to 100% reduction in tumor volume was observed within 3 to 4 days posttreatment. However, tumors relapsed within 5 to 13 days. Persistent, tumor regression was observed only when protocols using successive injections of combined immunotoxin/mafosfamid were used. All seven mice undergoing this treatment had a precipitous decrease in tumor size, and 86% survived greater than 30 days posttreatment. No residual tumor was detectable on Day 30 in five of seven mice. Regression was partly attributed to the selective activity of immunotoxin, since successive injections of an irrelevant control immunotoxin coupled to ricin in combination with mafosfamid did not reduce tumor size. Thus, we have demonstrated that a combination of anticancer chemotherapy and immunotoxin therapy yielded a greater antitumor effect than either therapy alone.

Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and leu19 antigens but negative expression of CD16 antigens.

The phenotype and function of lymphocytes from cancer patients treated with repetitive weekly cycles of continuous i.v. infusions of recombinant interleukin 2 (IL-2) were examined. Peripheral blood lymphocytes (PBL) obtained after IL-2 therapy showed an increased percentage of cells bearing the CD16 and leu19 markers which are associated with natural killer cells. These PBL mediated significantly increased levels of IL-2-dependent lymphokine-activated killer (LAK) activity against the Daudi cell line. Depletion of CD16+ cells from PBL obtained after in vivo IL-2 caused only slight inhibition of their LAK activity or their proliferative response to IL-2 in vitro. This indicates that CD16+ cells are involved but play only a minor role in these responses. In contrast, depletion of leu19+ cells, from PBL activated in vivo with IL-2, virtually abrogated their LAK activity and their proliferative response to IL-2. Two-color flow cytometry studies showed that a leu19+/CD16- population was expanded by in vivo IL-2 therapy and was responsible for the majority of LAK activity by in vivo-activated PBL. Moreover, this CD16- population showed an increased density of leu19 and CD2 (E rosette receptor) antigens when compared to the resting PBL obtained prior to IL-2 treatment. These data show that the predominant population mediating in vitro LAK activity, induced by in vivo IL-2 therapy, consists of activated natural killer cells with a high density of leu19 and CD2 antigens but negative for the CD16 antigen.

Repetitive weekly cycles of interleukin 2: effect of outpatient treatment with a lower dose of interleukin 2 on non-major histocompatibility complex-restricted killer activity.

Fifteen patients with advanced malignancy who had failed conventional therapy were entered into a protocol consisting of 1 inpatient mo of repetitive weekly cycles of interleukin 2 (IL-2) at 3 x 10(6) units/m2/day by constant infusion for the first 4 days of each week. This was followed by IL-2 administered on an outpatient basis at the same schedule but at a dose of 1 x 10(6) units/m2/day for the next 1 to 6 mo. Nine patients had renal carcinoma, four had melanoma, and two had lymphoma. Thirteen patients completed the induction month, and ten patients completed greater than or equal to 1 mo of outpatient therapy. Only one patient had therapy discontinued because of toxicity due to IL-2. No major toxicities occurred during outpatient therapy. After 1 mo of IL-2 at 3 x 10(6) units/m2/day, profound changes similar to those previously documented were seen in peripheral blood lymphocyte (PBL) counts (4.7-fold increase), lymphokine-activated killer activity (16-fold increase), and the percentage of PBL with natural killer-associated markers including a 3.6-fold increase in the percentage of PBL expressing the Leu 19 (NKH-1) marker, a 3.7-fold increase in Leu 11 (FcIgGR), and a 3.0-fold increase in Leu 17 (OKT10). These indicators of IL-2 effect all remained elevated relative to the baseline at the end of 1 and 2 mo of outpatient therapy at the lower dose. However, lymphokine-activated killer activity and Leu 17 percentage were significantly reduced relative to the effect of the higher induction dose. PBL taken from patients while receiving maintenance therapy showed strong and rapid responses to IL-2 in vitro, confirming the in vivo effects of prolonged IL-2 treatment. Nevertheless, there were no complete or partial antitumor responses seen. This study demonstrates that an immunologically active dose of IL-2 can be given long term as outpatient therapy with tolerable toxicity and results in highly IL-2-responsive "primed" lymphokine-activated killer cells.

Natural killer cells activated by interleukin 2 treatment in vivo respond to interleukin 2 primarily through the p75 receptor and maintain the p55 (TAC) negative phenotype.

Interleukin 2 (IL-2) induced activation of unstimulated resting natural killer (NK) cells or resting T-cells initially occurs following binding of IL-2 through the p75 receptor that is expressed primarily by these cells. However, this IL-2/p75 interaction induces TAC chain synthesis and formation of high affinity IL-2 receptor required for the proliferation of resting peripheral blood lymphocytes. In this study, we present data indicating that NK cells activated by in vivo IL-2 treatment, in contrast to resting NK cells, respond and proliferate to further IL-2 in vitro using primarily the p75 receptor with only a minor component of cells responding through the high affinity receptor. These in vivo activated NK cells minimally expressed the TAC chain and maintained this TAC negative phenotype while proliferating in response to IL-2. The primary involvement of the p75 receptor in the proliferative response of these cells to IL-2 was demonstrated by the need for concentrations of IL-2 higher than 44 pM to obtain a significant response and by the dramatic inhibition of this response by anti-p75 monoclonal antibody. Anti-TAC monoclonal antibody inhibited only the poor proliferation obtained at low doses of IL-2 suggesting a minor role for TAC and high affinity IL-2 receptors. This was in contrast to the partial inhibition of proliferation by anti-p75 or anti-TAC observed in unstimulated pretherapy peripheral blood lymphocytes suggesting that these cells respond to IL-2 through both high affinity receptors and intermediate affinity p75 receptors. The T-cells isolated from in vivo activated peripheral blood lymphocytes, despite expressing TAC, were not responsive to IL-2, suggesting that these cells express predominantly nonfunctional low affinity TAC receptors. NK cells activated by IL-2 in vivo represent a unique model system of IL-2 dependent cells that respond and proliferate to IL-2 essentially through the p75 IL-2 receptor.

In vivo induction of the lymphokine-activated killer phenomenon: interleukin 2-dependent human non-major histocompatibility complex-restricted cytotoxicity generated in vivo during administration of human recombinant interleukin 2.

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the immunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2 and enhanced direct cytotoxic activity on K562 target cells. This lytic activity was further augmented by the addition of IL-2 during the 51Cr release assay. Fresh peripheral blood lymphocytes from some patients who had just completed treatment at the higher IL-2 dose levels were able to kill both the natural killer-resistant Daudi cell line and fresh tumor cells while pretreatment samples and peripheral blood lymphocytes from healthy controls were not. This lytic activity was best detected when IL-2 was present in the in vitro effector assay. These results demonstrate that the administration of IL-2 to patients with cancer induces a population of effector cells able to directly destroy natural killer-resistant target cells, when assayed in the presence of IL-2.

The neuropharmacology of schistosomes.

The schistosomal nervous system uses some of the same neurotransmitters as the vertebrate system, but significant differences exist with respect to neurotransmitter metabolism and receptor specificity. The cholinergic system has been most extensively studied, and the serotoninergic system has also been examined carefully; catecholamines and benzodiazepines also seem to affect nervous system function. The objective of this work has been to determine whether neuroactive drugs could be used to disrupt the parasites' physiology sufficiently to have therapeutic impact, while avoiding toxic effects on the host nervous system. Promising findings have been made, suggesting that the unique properties of the schistosomal nervous system might be exploited to therapeutic advantage.

Phase II trial of sequential radiation and interleukin 2 in the treatment of patients with metastatic renal cell carcinoma.

We have previously demonstrated that local tumor irradiation effectively enhanced the therapeutic effect of interleukin 2 (IL-2) therapy in an experimental murine renal adenocarcinoma model. Based on these preclinical studies, we have designed and initiated a Phase II trial of irradiation combined with IL-2 for the treatment of metastatic renal cell carcinoma. Patients received 800 cGy to the primary or metastatic lesions on days 1 and 15 followed by IL-2 (600,000 IU/kg i.v.) every 8 h on days 4-8 and 18-22. Sixteen patients were entered; all completed treatment and are therefore evaluable for toxicity and response. Two partial remissions were seen for a response rate of 12.5% (95% confidence interval, 0-28.7). There was no increase in toxicity over that which is anticipated from IL-2 alone. The antitumor activity seen in this trial is consistent with what would be expected from high-dose IL-2 alone.

The mechanism of local tumor irradiation combined with interleukin 2 therapy in murine renal carcinoma: histological evaluation of pulmonary metastases.

We have demonstrated that tumor irradiation enhanced the therapeutic effect of interleukin 2 (IL-2) on pulmonary metastases from a murine renal adenocarcinoma, Renca. To investigate the mechanism of interaction between tumor irradiation and IL-2 therapy, we have histologically evaluated the effects of each therapy alone or in combination on Renca pulmonary metastases. Following treatment of established lung metastases with irradiation and IL-2 therapy, lung sections were processed for H&E or immunohistochemical staining. We found that tumor irradiation or IL-2 therapy locally induced vascular damage, resulting in multifocal hemorrhages and mononuclear cell mobilization in the lung tissue. This effect was amplified in lungs treated with the combined therapy. Immunohistochemistry showed that irradiation produced a macrophage influx into irradiated tumor nodules, and systemic IL-2 therapy induced T-cell infiltration in tumor nodules. Lungs treated with the combined therapy exhibited massive macrophage, T-cell, and natural killer cell mobilization in disintegrating tumor nodules and in the lung tissue. This combined therapy caused a decrease in the number of proliferating tumor cells and an increase in the number of apoptotic cells, which were more marked than with either therapy alone. We suggest that the macrophages mobilized by radiation-induced tissue injury could play a role in phagocytosis of apoptotic tumor cells, processing and presenting of tumor antigens for a systemic immune response activated by IL-2. Tumor destruction may result from the concomitant action of activated T cells, natural killer cells, and macrophages infiltrating the tumor nodules.

Systemic interleukin 2 therapy for human prostate tumors in a nude mouse model.

Once the regional lymph nodes become involved in prostate carcinoma, 85% of patients develop distant metastases within 5 years, and metastatic disease is difficult to treat. We have investigated the effect of systemic interleukin 2 (IL-2) treatment on metastatic prostate carcinoma using a xenograft tumor model. Cells from a PC-3/IF cell line, produced by intrafemoral injection of human PC-3 prostate carcinoma cells, were injected in the prostate of Balb/c nude mice. Prostate tumors and para-aortic lymph nodes were resected, and tumor cells were recultured and passaged in the prostate in vivo to produce new cell lines. On day 6 following prostatic injection of these cell lines, mice were treated with i.p. injections of IL-2 at 25,000-50,000 units/ day for 5 consecutive days. The effect of IL-2 on tumor progression was assessed, and histological studies were performed on prostate tumor and lymph node sections. The tumor cell lines generated by serial prostate injection were tumorigenic and metastasized to regional para-aortic lymph nodes. Tumors of 0.4 cm were obtained by day 16 and grew to 1-1.5 cm by day 40 with metastasis to para-aortic lymph nodes. Following two to three weekly courses of 5 days of 25,000-40,000 units/day of IL-2, the growth of prostate tumors was inhibited by 94%. Higher doses of 50,000 units/ day were toxic. Histologically, prostate sections showed vascular damage manifested by multifocal hemorrhages and an influx of lymphocytes and polymorphonuclear cells into disintegrating tumors and areas of necrosis containing numerous apoptotic cells. In contrast to control mice, para-aortic lymph nodes were not enlarged in responding mice. These findings suggest that systemic IL-2 therapy can induce an antitumor response in prostate tumors and control their growth and metastasis.

Neutron or photon irradiation for prostate tumors: enhancement of cytokine therapy in a metastatic tumor model.

We have shown that implantation of human prostate carcinoma PC-3 cells in the prostates of nude mice led to the formation of prostate tumors with metastases to para-aortic lymph nodes. We found that day 6 prostate tumors were responsive to systemic injections of interleukin 2 (IL-2) therapy. We have now investigated the combination of primary tumor irradiation and IL-2 for metastatic prostate cancer in this preclinical tumor model. The effect of neutron radiation was compared with that of photon radiation. Advanced prostate tumors (approximately 0.4 cm) were irradiated, and a day later, mice were treated with systemic IL-2 for three weekly cycles. In separate experiments, mice were either sacrificed on day 30 to assess prostate tumor size and tumor histology or followed for survival. A dose-dependent inhibition of prostate tumor growth was caused either by photons or neutrons, but neutrons were more effective than photons with a relative biological effectiveness of 2. The tumor inhibition obtained with 250 cGy neutrons and 500 cGy photons was significant (>75%) and was further increased (> or = 90%) by addition of IL-2 therapy. In survival studies, the combination of radiation and IL-2 showed a significant survival advantage compared with untreated mice (P < or = 0.005) or radiation alone (P < or = 0.003) and an increase in median survival compared with IL-2 alone. Histologically, the combined regimen resulted in a greater degree of tumor destruction, inflammatory response, and vascular damage than that observed with each modality alone. After this combined treatment, no tumor was histologically detected in the para-aortic lymph nodes of these mice, and the lymph nodes were significantly smaller. These findings showed that primary tumor irradiation, either with neutrons or photons, enhanced IL-2 therapeutic effect for the treatment of advanced prostate cancer. This combined modality induced an antitumor response that controlled the growth of prostate tumors and their metastases.

Genistein potentiates the radiation effect on prostate carcinoma cells.

We have shown previously that genistein, the major isoflavone in soybean, inhibited the growth of human prostate cancer cells in vitro by affecting the cell cycle and inducing apoptosis. To augment the effect of radiation for prostate carcinoma, we have now tested the combination of genistein with photon and neutron radiation on prostate carcinoma cells in vitro. The effects of photon or neutron radiation alone or genistein alone or both combined were evaluated on DNA synthesis, cell growth, and cell ability to form colonies. We found that neutrons were more effective than photons for the killing of prostate carcinoma cells in vitro, resulting in a relative biological effectiveness of 2.6 when compared with photons. Genistein at 15 microM caused a significant inhibition in DNA synthesis, cell growth, and colony formation in the range of 40-60% and potentiated the effect of low doses of 200-300 cGy photon or 100-150 cGy neutron radiation. The effect of the combined treatment was more pronounced than with genistein or radiation alone. Our data indicate that genistein combined with radiation inhibits DNA synthesis, resulting in inhibition of cell division and growth. Genistein can augment the effect of neutrons at doses approximately 2-fold lower than photon doses required to observe the same efficacy. These studies suggest a potential of combining genistein with radiation for the treatment of localized prostate carcinoma.