Changes in circulating microRNA levels associated with prostate cancer.

BACKGROUND: The aim of this study was to investigate the hypothesis that changes in circulating microRNAs (miRs) represent potentially useful biomarkers for the diagnosis, staging and prediction of outcome in prostate cancer. METHODS: Real-time polymerase chain reaction analysis of 742 miRs was performed using plasma-derived circulating microvesicles of 78 prostate cancer patients and 28 normal control individuals to identify differentially quantified miRs. RESULTS: A total of 12 miRs were differentially quantified in prostate cancer patients compared with controls, including 9 in patients without metastases. In all, 11 miRs were present in significantly greater amounts in prostate cancer patients with metastases compared with those without metastases. The association of miR-141 and miR-375 with metastatic prostate cancer was confirmed using serum-derived exosomes and microvesicles in a separate cohort of patients with recurrent or non-recurrent disease following radical prostatectomy. An analysis of five selected miRs in urine samples found that miR-107 and miR-574-3p were quantified at significantly higher concentrations in the urine of men with prostate cancer compared with controls. CONCLUSION: These observations suggest that changes in miR concentration in prostate cancer patients may be identified by analysing various body fluids. Moreover, circulating miRs may be used to diagnose and stage prostate cancer.

Dasatinib inhibits the growth of prostate cancer in bone and provides additional protection from osteolysis.

BACKGROUND: Dasatinib is a small molecule kinase inhibitor that has recently been shown to inhibit Src family kinases (SFK) and also has activity against CaP. Of importance to metastatic CaP, which frequently metastasises to bone, SFK are also vital to the regulation of bone remodelling. We sought to determine the ability of dasatinib to inhibit growth of CaP in bone. METHODS: C4-2B CaP cells were injected into tibiae of SCID mice and treated with dasatinib, alone or in combination with docetaxel. Serum prostate-specific antigen levels, bone mineral density, radiographs and histology were analysed. RESULTS: Treatment with dasatinib alone significantly lowered sacrifice serum prostate-specific antigen levels compared to control, 2.3+/-0.4 vs 9.2+/-2.1 (P=0.004). Combination therapy improved efficacy over dasatinib alone (P=0.010). Dasatinib increased bone mineral density in tumoured tibiae by 25% over control tumoured tibiae (P<0.001). CONCLUSION: Dasatinib inhibits growth of C4-2B cells in bone with improved efficacy when combined with docetaxel. Additionally, dasatinib inhibits osteolysis associated with CaP. These data support further study of dasatinib in clinical trials for men with CaP bone metastases.

Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer.

The gene for E3 ubiquitin ligase WWP1 is located at 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that WWP1 negatively regulates the TGFbeta tumor suppressor pathway by inactivating its molecular components, including Smad2, Smad4 and TbetaR1. These findings suggest an oncogenic role of WWP1 in carcinogenesis, but direct supporting evidence has been lacking. In this study, we examined WWP1 for gene dosage, mRNA expression, mutation and functions in a number of human prostate cancer samples. We found that the WWP1 gene had copy number gain in 15 of 34 (44%) xenografts and cell lines from prostate cancer and 15 of 49 (31%) clinical prostate cancer samples. Consistently, WWP1 was overexpressed in 60% of xenografts and cell lines from prostate cancer. Mutation of WWP1 occurred infrequently in prostate cancer. Functionally, WWP1 overexpression promoted colony formation in the 22Rv1 prostate cancer cell line. In PC-3 prostate cancer cells, WWP1 knockdown significantly suppressed cell proliferation and enhanced TGFbeta-mediated growth inhibition. These findings suggest that WWP1 is an oncogene that undergoes genomic amplification at 8q21 in human prostate cancer, and WWP1 overexpression is a common mechanism involved in the inactivation of TGFbeta function in human cancer.

Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer.

The expression level of the androgen receptor (AR) gene in androgen-dependent and -independent prostate cancer was determined by using real-time quantitative reverse transcription-PCR assay. Eight benign prostate hyperplasias, 33 untreated and 13 hormone-refractory locally recurrent carcinomas, as well as 10 prostate cancer xenografts, were analyzed. All hormone-refractory tumors expressed AR and showed, on average, 6-fold higher expression than androgen-dependent tumors or benign prostate hyperplasias (P < 0.001). Four of 13 (31%) hormone-refractory tumors contained AR gene amplification detected by fluorescence in situ hybridization. Androgen-independent tumors with gene amplification expressed, on average, a 2-fold higher level of AR than the refractory tumors without the gene amplification. Two xenografts (LuCaP 35 and 69) showed amplification and high-level expression of the AR gene. These xenografts are the first prostate cancer model systems containing the gene amplification. The findings demonstrate that AR is highly expressed in androgen-independent prostate cancer, suggesting that the AR signaling pathway is important in the progression of prostate cancer during endocrine treatment. The two xenografts with the AR gene amplification will enable studies evaluating the functional significance of the amplification and development of new treatment strategies based on high-level expression of AR.

Placental alkaline phosphatase as a tumor marker for seminoma.

A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.

Correlation of vascular permeability and blood flow with monoclonal antibody uptake by human Clouser and renal cell xenografts.

The specific uptake of 125I-A6H antibody by xenografts of the human renal cell carcinoma (RCC) TK177G in the athymic mouse was considerably greater than that seen for other human tumor xenografts and their associated antibodies (e.g., 125I-B6.2 uptake by the human breast carcinoma, Clouser). In addition the A6H-RCC model also demonstrated both greater localization indices and absolute amount of antibody bound than did the B6.2-Clouser model. Several physiological factors were studied to assess whether they might play a role in this greater specific uptake. Vascular volume was determined using the in situ labeling of red blood cells with 99mTc. Vascular permeability was determined by measuring the amount of 125I-labeled bovine serum albumin and 131I-labeled nonspecific IgG1 (anti-horseradish peroxidase) extravasated out of the tumor vasculature during 1 hr. Relative blood flow to the tumor was determined using the 86Rb method. Blood flow and vascular permeability were found to be significantly greater in the RCC tumor xenografts than in Clouser tumors. Differences in vascular permeability were especially dramatic, showing the vasculature of the RCC xenograft was twice as permeable as that of the Clouser tumor. Animals bearing either RCC or Clouser xenografts were injected with a monoclonal antibody to human major histocompatibility complexes (125I-labeled anti-human histocompatibility complex A, B, C). Tumor uptake of 125I-labeled anti-human histocompatibility complex A, B, C was found to be 5 times greater in RCC than Clouser xenografts. These results, therefore, suggest that the differences seen in the physiological factors studied can account for some of the greater specific 125I-A6H uptake by the RCC tumor than 125I-B6.2 uptake by the Clouser xenograft.

Localization of human renal cell carcinoma xenografts with a tumor-preferential monoclonal antibody.

We previously described an immunoglobulin G1 monoclonal antibody (UMVA-RCC-A6H) that is highly reactive with human renal cell carcinoma (RCC) and has little cross-reactivity to other cell types both normal and malignant. In efforts detailed herein, radiolabeled A6H selectively localized to RCC xenografts and provided high resolution images of the xenografts. Also, A6H clearly discriminated between RCC xenografts and other human tumor xenografts. Consistent images of RCC xenografts (greater than 60 mg) were obtained without background subtraction. The amount of radiolabeled A6H in the tumor usually ranged from five to twenty times that of the blood. Normal mouse tissues, abscesses, and other human tumor xenografts contained less radiolabel per mg than did blood. A control monoclonal antibody of the same isotype failed to exhibit any localization in xenografts or normal tissues. Approximately 40% of the radiolabeled A6H dose per g was localized in the RCC xenograft 2 days after injection, although at the time of imaging about 60% of the radiolabel remaining in the mouse was associated with the xenograft. These results demonstrate that a RCC restrictive monoclonal antibody does specifically localize to RCC xenografts and supports the hope that this approach may have clinical value for diagnosis, staging, or treatment.

Monoclonal antibodies to human renal cell carcinoma: recognition of shared and restricted tissue antigens.

Monoclonal antibodies (MABs) reactive with human renal cell carcinoma (RCC) were generated following immunization of mice with either RCC homogenates, RCC cell lines, or fetal kidney homogenates. The characteristics of four highly reactive immunoglobulin G1 MABs, designated UMVA-RCC-A6H, UMVA-RCC-A36, UMVA-RCC-C5H and UMVA-RCC-D5D are presented. The screening process consisted of a cell binding enzyme-linked immunosorbent assay and immunohistological examination of tumor, normal, and fetal tissue sections. The MABs illustrated various degrees of antigen restriction: A6H identified an antigen common to RCC, some lung and colon carcinomas, the proximal renal tubules but no other normal tissues; A36 reacted with most human tumors, the renal tubules, and many other normal tissues; C5H reacted with nearly every human cancer but of the normal tissues, only the renal glomerulus shared this antigen; D5D was very restrictive, reacting with many although not all RCC and no other cancers or normal tissues with the exception of an occasional reactivity with a Bowman's capsule. Metastatic RCC and RCC xenografts expressed these antigens. None of the MABs participated in complement-mediated cytotoxicity. In immunoprecipitation studies with L-[methyl-3H]methionine and [3H]glucosamine-HCl metabolically labeled RCC cells, C5H was shown to be associated with an antigen of Mr 115,000.

Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Comparison of Fc-osteoprotegerin and zoledronic acid activities suggests that zoledronic acid inhibits prostate cancer in bone by indirect mechanisms.

Zoledronic acid (ZA) has been shown to inhibit prostate tumor growth in vitro and have beneficial effects in patients with advanced prostate cancer (CaP). The aim of this study was to determine whether ZA exhibits direct anti-tumor effects on CaP cells in vivo. To distinguish the effects of inhibition of osteolysis and direct anti-tumor activity of ZA in vivo, we compared the results of treatment with ZA and osteoprotegerin (Fc-OPG), which inhibits osteolysis, but without significant direct anti-tumor effects. In vitro Fc-OPG had no significant effects on C4-2 proliferation, whereas ZA decreased proliferation. However, both agents decreased tumor growth in bone. Moreover, both increased bone volume and prevented the overall decreases in BMD associated with growth of C4-2 cells in bone. Our study provides novel and significant observations that the in vivo effects of ZA are consistent with indirect effects mediated by osteoclasts.

Differential expression of osteonectin/SPARC during human prostate cancer progression.

The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP.

Early tumor cell dissemination in patients with clinically localized carcinoma of the prostate.

Because a significant number of patients with pathologically organ-confined carcinoma of the prostate subsequently develop recurrent disease, metastasis may occur much earlier than previously believed. We have used a reverse transcription-PCR assay for prostate-specific antigen mRNA and an immunocytochemical staining method for cytokeratins to test this hypothesis in paired peripheral blood (PB) and bone marrow (BM) specimens from 71 patients with clinically localized disease before radical prostatectomy, 14 patients with advanced-stage carcinoma of the prostate, and 30 controls (young healthy volunteers, patients without prostate disease, and patients with benign prostatic hyperplasia). Controls were negative in BM and PB. Fifty-six% of patients with organ-confined tumors (pT2) and 73% of those with extracapsular extension (pT3) were positive in the BM versus 16% of those with pT2 tumors and 27% of those with pT3 tumors in the PB. Patients with advanced-stage disease were positive in 86% of BM versus 71% of PB. The sensitivity of the immunocytochemistry assay to detect tumor cells was lower as compared with the reverse transcription-PCR assay. The results suggest that tumor cell dissemination occurs early during disease progression. Prostate cells seem to preferentially concentrate in the BM rather than the PB, which may be due to sequestration there by homing mechanisms. As the rate of detection in the BM exceeds the proportion of patients with subsequently progressing disease, we hypothesize that only a subset of these cells can survive in the BM and evolve to clinically apparent disease.

Characterization of a novel androgen-sensitive, prostate-specific antigen-producing prostatic carcinoma xenograft: LuCaP 23.

Prostatic carcinoma has proven extremely difficult to establish as cell lines or xenografts. In this article, we describe a new series of prostate cancer xenografts propagated in athymic mice, designated LuCaP 23, developed from prostate metastases harvested at autopsy shortly after death. Tumor from three separate metastatic deposits was developed into three xenograft sublines: two from lymph node metastases (LuCaP 23.1 and 23.8) and one from a liver metastasis (LuCaP 23.12). Fluorescence in situ hybridization analysis confirms the xenografts are human. Histologically, the xenografts are comprised of columnar epithelial cells arranged in a glandular pattern. Tumor doubling times range from 11 to 21 days for the three sublines. The cells secrete large amounts of prostate-specific antigen (PSA) with PSA indices of 1.27, 1.63, and 5.21 ng/ml/mm3 for the mice bearing the LuCaP 23.1, 23.8, and 23.12 sublines, respectively. Following androgen deprivation a temporary decrease in PSA secretion and a decrease in tumor size are noted in most tumors. Eventually, the tumors become androgen independent and resume growth in castrate hosts. The degree of PSA response to castration and time to PSA nadir correlate with time to progression. Thus, unlike most existing models of prostatic carcinoma, this novel xenograft exhibits many phenotypic characteristics of clinical prostatic carcinoma, including androgen sensitivity. These properties make this xenograft an excellent model for future study.

Serum osteoprotegerin levels are increased in patients with advanced prostate cancer.

PURPOSE: Osteoprotegerin (OPG) is a soluble osteoclastogenesis inhibitor that regulates bone turnover. We reported recently that OPG protein expression is significantly increased in prostate cancer (CaP) cells present in bone metastases. The aim of this study was to determine serum OPG levels in patients at different stages of CaP and correlate the results with disease status. EXPERIMENTAL DESIGN: OPG levels were examined in patients with benign prostatic hyperplasia, clinically localized CaP, early recurrence of CaP, and advanced CaP and evidence of bone metastases. Serum OPG levels were measured by sandwich ELISA assays. The serum Crosslaps (sCTX) assay was used to quantify bone resorption in the advanced CaP group. RESULTS: Serum OPG levels were increased significantly in the advanced CaP group versus all other groups. There was no significant correlation between serum OPG levels and PSA levels either in the advanced CaP group or within any of three treatment subclasses of this group: no Tx, those not treated; Tx, those treated; and R, those treated with resorption blockers. Levels of OPG were negatively correlated with sCTX levels only in the advanced CaP Tx group. sCTX levels correlated with prostate-specific antigen levels in the advanced CaP Tx and R groups but not in the no-Tx group. CONCLUSIONS: Our data show that serum OPG levels are increased with advanced CaP. We hypothesize that OPG levels are related to CaP progression and suggest that further studies of the biological effects of OPG on CaP metastases are warranted.

Cathepsin K mRNA and protein expression in prostate cancer progression.

Prostate cancer (CaP) is the most commonly diagnosed malignancy in men and is often associated with bone metastases, which cause much of the morbidity associated with CaP. Lesions associated with CaP generally exhibit increased bone formation and resorption. Increased bone resorption may release factors from the extracellular matrix that contribute to tumor growth. Cathepsin K (cat K) is a cysteine protease that exhibits strong degradative activity against the extracellular matrix and is involved in osteoclast-mediated bone destruction. In this study, we analyzed the expression of cat K in CaP cell lines and patient samples. Cat K message was detected in CaP cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and in primary CaP and metastases by in situ hybridization. Immunohistochemistry revealed variable expression of cat K in primary CaP samples, as well as nonosseous metastases, whereas expression in bone metastases was significantly higher than in primary CaP, and normal prostate tissues were negative. Cat K protein was detected in CaP cell lines by Western blotting after immunoprecipitation. Cat K enzymatic activity was also detected in CaP cell lines by a fluorogenic assay and by an assay for degradation of collagen type I. Increased levels of NTx, a marker of bone matrix degradation mediated primarily by cat K, were also detected in sera of patients with CaP bone metastases. We hypothesize that CaP-expressed cat K may contribute to the invasive potential of CaP, while increased expression in bone metastases is consistent with a role in matrix degradation.

High prevalence of antibodies against HERV-K10 in patients with testicular cancer but not with AIDS.

Human endogenous retrovirus K10 (HERV-K10) env and gag expression has been detected in placenta, embryonic tissue, and cell lines. By transfection, these sequences have been expressed in insect cells and developed into serological assays, revealing HERV-K10 antibodies in patients with testicular cancer. Patients with AIDS are at an increased risk for testicular cancer and frequently reactivate latent infections. We postulated that HERV-K10 seroprevalence might be increased with HIV infection or AIDS. Stored, frozen serum samples from 52 patients with testicular cancer (8 patients with HIV and 30 patients with samples near the time of diagnosis) and 84 controls (40 patients with HIV) were diluted 1:40 and tested by immunofluorescence against SF158 cells transfected with HERV-K10 env [ENV1.9(+)] or gag (pACGAG). Seroprevalence rates were compared cross-sectionally in cases and controls, excluding those with indeterminate results (3 of 30 cases and 7 of 84 controls), and also were examined longitudinally in the cases before or after diagnosis of testicular cancer. Seroprevalence to HERV-K10 Env or Gag was 17 of 27 testicular cancer patients (63%) around the time of diagnosis, compared to 4 of 77 controls (5%; P < 0.0001). Seroprevalence was similar (50% to 60%) with seminoma, teratocarcinoma, or embryonal carcinoma, and it was not increased with HIV infection in either cases (33%) or controls (3%). HERV-K10 antibodies were detected in 12 of 19 cases (63%) more than 6 months before seminoma diagnosis, as well as in four cases with residual or recurrent malignancy more than 1 month after initial diagnosis. Thus, HERV-K10 antibodies are detected frequently with testicular cancer and seem to resolve rapidly with effective therapy of the malignancy. Antibody reactivity also occurs in approximately 5% of controls, perhaps because of nonspecific or cross-reactive epitopes. HIV and AIDS were not associated with HERV-K10 antibodies, thus, leaving their higher risk of testicular cancer unexplained.

Bone histology at autopsy and matched bone scintigraphy findings in patients with hormone refractory prostate cancer: the effect of bisphosphonate therapy on bone scintigraphy results.

Bisphosphonates (BisP) are non-metabolized compounds with high bone affinity used in bone metastasis diagnosis and treatment. Currently, BisP are used to treat hypercalcemia of malignancy as well as to prevent, minimize, or delay skeletal morbidity. These compounds have a long half-life in bone. Thus long-term BisP treatment might saturate bone and interfere with a single-dose scanning agent used for bone scintigraphy when visualizing bone metastases. In an effort to answer this question, this study evaluated the concordance of histology and Technetium99 methylene diophosphonate (Tc99 MDP) bone scintigraphy in the diagnosis of bone metastases in prostate cancer patients. We assessed the concordance of findings between bone scintigraphy and histology using 188 bone biopsies from 11 autopsied patients who died with metastatic prostate cancer, 5 of whom were treated with pamidronate for 2 to 13 months before death. Overall agreement between histology and bone scintigraphy was 84%, 86% in non-pamidronate-treated patients and 82% in pamidronate-treated patients. Scintigraphic bone metastases without histological metastasis (false negatives = 12.7%) were observed in 24 anatomic locations; half of these were in one patient who had been treated with pamidronate and had no histological bone response to the carcinoma. There were only 4 sites where a positive bone scan was not associated with histologic metastasis (false positives = 2.21%). There was no statistical difference between the treated and non-treated group for concordance, specificity, sensitivity, positive and negative predictive values of bone scintigraphy and prevalence of histological abnormality. Long-term pamidronate treatment of prostate cancer bone metastases does not generally affect the ability to detect bone metastases with Tc99 MDP bone scintigraphy.

The effect of osteoprotegerin administration on the intra-tibial growth of the osteoblastic LuCaP 23.1 prostate cancer xenograft.

Osteoprotegerin (OPG) plays a central role in controlling bone resorption. Exogenous administration of OPG has been shown to be effective in preventing osteolysis and limiting the growth of osteolytic metastasis. The objective of this study was to investigate the effects of OPG on osteoblastic prostate cancer (CaP) metastases in an animal model. LuCaP 23.1 cells were injected intra-tibially and Fc-OPG (6.0 mg/kg) was administered subcutaneously three times a week starting either 24 hours prior to cell injection (prevention regimen) or at 4 weeks post-injection (treatment regimen). Changes in bone mineral density at the tumor site were determined by dual x-ray absorptiometry. Tumor growth was monitored by evaluating serum prostate specific antigen (PSA). Fc-OPG did not inhibit establishment of osteoblastic bone lesions of LuCaP 23.1, but it decreased growth of the tumor cells, as determined by decreases in serum PSA levels of 73.0 +/- 44.3% (P < 0.001) and 78.3 +/- 25.3% (P < 0.001) under the treatment and prevention regimens, respectively, compared to the untreated tumor-bearing animals. Administration of Fc-OPG decreased the proliferative index by 35.0% (P = 0.1838) in the treatment group, and 75.2% (P = 0.0358) in the prevention group. The results of this study suggest a potential role for OPG in the treatment of established osteoblastic CaP bone metastases.

Newer applications of serum prostate-specific antigen in the management of prostate cancer.

The information contained in this article indicates that PSA will have an increasing role in the management of prostate cancer. For example, it is now essential for optimal diagnosis if prostate cancer detection is the goal. Prospects are high that more information about PSA density relationships, PSA velocity phenomenologies, and possible PSA isoforms will increase diagnostic accuracy. It would also seem that PSA will improve staging accuracy not only by better manipulation of multiple preoperative parameters (eg, cancer grade, volume, PSA, etc) but possibly by the molecular detection of minute amounts of occult prostate cancer cells in bone, blood or lymph nodes, or by improved use of immune scanning. Finally, the use of these more sophisticated staging approaches together with increasingly sensitive PSA assays and possibly androgen provocative testing might allow the prospect that the potentially curative therapies can be almost immediately assessed for efficacy, thereby increasing prospects for therapeutic progress. Finally, PSA may become even more important for manipulating hormone therapies (eg, IAS therapy) or it could form a basis for new treatments such as immune or gene therapy.

A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis.

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.