Quantification of allyl hexanoate in pineapple beverages and yogurts as a case study to characterise a source of uncertainty in dietary exposure assessment to flavouring substances.

One source of uncertainty in the estimation of dietary exposure to flavouring substances is the uncertainty in the occurrence and concentration levels of these substances naturally present or added to foodstuffs. The aim of this study was to assess the variability of concentration levels of allyl hexanoate, considered as a case study, in two main food categories to which it is often added: pineapple juice-based beverages and yogurts containing pineapple. Thirty-four beverages and 29 yogurts, with pineapple fruit or juice and added flavourings declared as ingredients on the package, were purchased from the local market (in Rome) and analysed. Analytical methods based on the stir bar sorptive extraction (SBSE) technique for the isolation of the target analyte, and on GC-MS analysis for final determination, were developed for the two food categories. In beverages, allyl hexanoate concentrations ranged from less than 0.01 to 16.71 mg l(-1), whereas in yogurts they ranged from 0.02 to 89.41 mg kg(-1). Average concentrations in beverages and yogurts with pineapple as the main fruit ingredient (1.91 mg l(-1) for beverages, 9.61 mg kg(-1) for yogurts) were in fair agreement with average use level data reported from industry surveys for the relevant food categories (4.5 and 6.0 mg kg(-1), respectively). Within the group of yogurts a single product was found to contain a level of allyl hexanoate more than 10-fold higher than the average reported use level. The screening techniques developed by the European Food Safety Authority (EFSA) using use level data provided by industry gave estimates of exposure that were of the same order of magnitude as the estimates obtained for regular consumers who would be loyal to the pineapple yogurt and beverage products containing the highest observed concentration of the substance of interest. In this specific case the uncertainty in the results obtained with the use of standard screening techniques for exposure assessment based on industry reported use levels is low.

Dietary exposure to flavouring substances: from screening methods to detailed assessments using food consumption data collected with EPIC-Soft software.

This study aimed to compare different methods of assessing dietary exposure to flavourings in the context of a stepwise approach. The dietary exposure to four flavourings--raspberry ketone, glycyrrhizinic acid, coumarin, and caffeine--was determined. When dietary exposure exceeded the safety limits, the need for more detailed assessment using less aggregated data was judged necessary. First, screening methods--maximized survey-derived daily intake (MSDI), single-portion exposure technique (SPET), and modified theoretical added maximum daily intake (mTAMDI)--were applied. Next, individual food consumption data were used for creating models with different levels of detail to identify the foods: a model based on food groups and models based on food items. These were collected from 121 Dutch adults using a standardized 2 x 24-h dietary recall (EPIC-Soft) in the European Food Consumption Validation (EFCOVAL) study. Three food item models were developed: without improvements of the flavouring descriptor built in the software; with improvements; and with use of non-specified flavour descriptors. Based on the results of at least one of the three screening methods, refined assessment was necessary for raspberry ketone, glycyrrhizinic acid, and caffeine. When applying the food group model, the need for refinement was indicated for the four flavourings. When applying the food item models, only glycyrrhizinic acid and caffeine presented dietary exposure above the safety limits. In the raspberry ketone case, dietary exposure increased when improvements in food description were considered. The use of non-specified flavour descriptors hardly changed the results. The collection of detailed food consumption data at the individual level is useful in the dietary exposure assessment of these flavourings.

Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells.

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12-26 (PPLSQETFSDLWKLL), residues 12-20, and 17-26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding alpha-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an alpha-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and waf(p21). Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

Chemosensitization of bladder carcinoma cells by bcl-xL antisense oligonucleotides.

PURPOSE: We investigated antisense inhibition of anti-apoptotic bcl-xL and bcl-2 proteins to increase chemosensitization in the T24 and 5637 bladder carcinoma cell lines. MATERIALS AND METHODS: A T24 bladder carcinoma cell line stably over expressing bcl-xL protein was constructed. Apoptosis by cytotoxic agents was estimated by cell cycle analysis and Annexin V binding. To eliminate bcl-xL expression T24 and 5637 cells were treated with C5-propynylated and 2'-O-methylribo-oligonucleotides. Levels of protein and messenger RNA were measured by Western and Northern blot analysis. Cell viability after combined treatment with oligonucleotides and various cytotoxic agents was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and evaluated statistically by Student's 2-sample t test. RESULTS: Forced over expression of bcl-xL protein desensitized the T24 bladder carcinoma cell line to cytotoxic agents. C5-propynylated and 2'-O-methylribo-oligonucleotides down-regulated bcl-xL protein expression in the T24 and 5637 cell lines, and increased their sensitivity to cytotoxic agents. The efficiency of antisense down-regulation of bcl-xL protein expression depended on the type of delivery agent. CONCLUSIONS: Antisense down-regulation of bcl-xL protein sensitizes bladder carcinoma cells to cytotoxic agents. However, it is possible that cellular chemosensitization results from a combination of effects, including nonsequence specificity, irrelevant cleavage and effects of the carriers combined with the specific antisense effects.

Formation of nuclear Bax/p53 complexes is associated with chemotherapy induced apoptosis.

Mechanisms by which chemotherapeutic agents induce apoptosis are not completely understood. Current knowledge of the actual pharmacologic effects of chemotherapy and their biochemical mechanisms are better understood than the downstream events, which initiate the apoptotic cascade. The chemotherapeutic agent cisplatin causes DNA damage and can induce apoptosis in several types of human cancers. We found the formation of previously unreported nuclear complexes between the tumor suppressor protein p53 and the pro-apoptotic protein Bax, in human melanoma cell lines induced into apoptosis following cisplatin exposure. These detergent resistant complexes were detected: after wild type (wt) p53 and Bax increased in the nucleus; at the same time when active cytoplasmic apoptosis related protease, caspase 3/CPP32 appeared; and prior to the detection of apoptotic DNA fragmentation. Three channel fluorescence laser scanning confocal image microscopy revealed that the nuclear Bax/p53 complexes remained in the nucleus and localized proximal to DNA fragmentation sites as assayed by TUNEL after cisplatin exposure. Two human melanoma cell lines, expressing wt p53, were induced into apoptosis after cisplatin exposure, however they differed in the timing of this induction. In both cell lines the formation of nuclear Bax/p53 co-immunoprecipitable complexes correlated with the timing of the induction of apoptosis. The degree of apoptosis induced by different concentrations of cisplatin correlated with the amount of nuclear Bax/p53 complexes. The co-immunoprecipitation of Bax and p53 was found regardless of the antibodies tested and was specific since Bcl-xL/p53 complexes were not detected. Additionally, the human prostate cancer cell line, LNCaP, also formed nuclear Bax/p53 complexes only after apoptosis was induced by paclitaxel.

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells.

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.

A study of the effect of energy healing on in vitro tumor cell proliferation.

This study examined the effect of energy healing on in vitro tumor cell growth using the cell culture model similar to that embraced by oncologists to assess the effect of chemotherapeutic agents. After selecting an energy healer based on his ability to influence this model, we assessed the effects of energy treatment compared to cells left at ambient temperature and to a control treatment consisting of a medical student mimicking the healer. A chi-square test comparing a medical student's and the practitioner's ability to inhibit tumor cell growth by 15% associates our practitioner with inhibition of tumor cell proliferation (p = 0.02). We also found that the magnitude of change was too close to the assay's intrinsic margin of error, thus making our quantitative data difficult to interpret. Although energy healing appears to influence several indices of growth in in vitro tumor cell proliferation, these assays are limited in their ability to define and prove the existence of this phenomenon. More sensitive biological assays are needed for further study in this field.

Evaluation of performance of the RIBA processor system for automated analysis of the strip immunoblot assay for detection of antibodies to hepatitis C virus.

The performance of a new automated analyzer for the processing and interpretation of the RIBA Strip Immunoblot Assay (SIA), used in the diagnosis of hepatitis C virus (HCV) infection, was evaluated. Laboratory performance of the RIBA SIA was compared with that of two manually processed supplementary anti-HCV tests (RIBA HCV 3.0 SIA and INNO-LIA HCV Antibody III). Specificity of the automated processing of SIA was 100% for 90 selected anti-HCV-negative samples. On the other hand, 119 of 120 (99.2%) previously confirmed anti-HCV-positive samples were also positive when assayed on the automated processor. Results for all specimens except one (51 of 52) were concordant for manual and automated RIBA, while 15 of 68 sera tested with automated RIBA and the INNO-LIA assay showed different patterns of reactivity. Three HCV sensitivity panels and one seroconversion panel were also compared. The results show a high sensitivity for SIA NS3- and NS5-encoded antigens. Moreover, data obtained for the anti-HCV seroconversion panel and for samples with borderline or discordant anti-HCV enzyme-linked immunosorbent assay results suggest that bands with a relative intensity of >0.5 on the automated analyzer (theoretically negative) should be evaluated with care. Coefficients of variability ranged from 9 to 14.8% in an interassay reproducibility study. Overall, the performance of the automated analysis of SIA is comparable to that of the manual RIBA assay. The new automated processor for SIA bands proved to be sensitive and specific. Its use makes the optical scoring of bands unnecessary by indicating relative intensity values, which could be particularly useful in the follow-up care of anti-HCV-positive patients receiving antiviral therapy.

Apoptosis in the rat penis after penile denervation.

PURPOSE: Despite the advances in nerve sparing prostatectomy for prostate cancer, some patients develop impotence or subjectively complain of a decrease in penile size. We hypothesized that these clinical observations may be explained by injury to the cavernous nerves resulting in programmed cell death (apoptosis) within the penis. We utilized a rat model of penile denervation in order to demonstrate apoptosis after denervation. METHODS AND MATERIALS: Fifteen male Sprague Dawley rats underwent abdominal exploration and bilateral cavernous neurotomy. Fifteen sham operations were performed as normal controls. The rats were sacrificed on postoperative day 1,2,3,6, and 10 and their penises were harvested. Messenger RNA was extracted and probed on a northern blot for sulfated glycoprotein-2 (SGP-2). SGP-2 is a gene product reported to be elevated in apoptotic tissues. Separate denervated and sham rats were used for DNA extraction (sacrificed postoperative day #2) in order to demonstrate the internucleosomal DNA fragmentation (laddering) found in apoptotic tissues. In addition, in situ histology was performed with ISEL techniques (in situ end labeling) to stain for apoptotic nuclei in denervated rats. RESULTS: Northern blot analysis showed a large increase in SGP-2 mRNA expression in the denervated rats with little detected in the sham operated group. DNA extraction studies revealed the presence of internucleosomal DNA fragmentation on agarose gel (a marker for apoptosis) in the denervated group versus intact high molecular weight DNA in the sham rats. In addition, in situ staining of denervated penile erectile tissue demonstrated apoptotic nuclei in the cavernous tissue. CONCLUSION: Apoptosis of penile erectile tissue occurs after denervation of the rat penis. This has not been previously described in the literature and may offer some explanation at the molecular level concerning the mechanism of impotence and/or decrease in penile size after radical prostatectomy.

Analytical and laboratory evaluation of a new fully-automated third generation enzyme immunoassay for the detection of antibodies to the hepatitis C virus.

An analytical and laboratory evaluation of a newly-developed fully-automated third generation ELISA for the detection of anti-HCV (Cobas Core Anti-HCV EIA, Roche) was undertaken. Coefficients of variation (CVs) calculated on positive control and serum samples ranged from 5.9 to 9.8% in the intra-assay precision test and from 3.9 to 11.3% in the inter-assay evaluation. With regard to the study of clinical laboratory performance, five groups of sera pre-screened with two third generation ELISA (Ortho HCV 3.0 ELISA; Innotest HCV Ab III) were assayed: anti-HCV negative samples (n = 932); anti-HCV positive samples (n = 449); difficult sera of different origin (n = 113); sera with discrepant results in the two ELISAs (n = 50); sera with an indeterminate result in one or more confirmatory test (n = 34). The overall concordance between the Roche anti-HCV EIA and the two reference assays was 97.5 and 97.8% for the Ortho and for the Innogenetics assays, respectively. Although it is not possible to provide absolute figures for clinical sensitivity and specificity, the results of the study on discrepant samples show that the Cobas Core Anti-HCV gives a number of negative results with positive or indeterminate confirmatory anti-HCV tests, which is intermediate between the Ortho and the Innogenetics assay. In contrast, only 5% Cobas Core Anti-HCV reactive sera are not positive or clear-cut single band reactive by supplemental assays. The results show that the new fully-automated third generation anti-HCV test is a valid alternative to other commercially available assays for screening of antibodies to the hepatitis C virus.

Preoperative reverse transcriptase polymerase chain reaction for prostate specific antigen predicts treatment failure following radical prostatectomy.

PURPOSE: We previously demonstrated than an enhanced reverse transcriptase-polymerase chain reaction assay for prostate specific antigen (PSA) can predict final pathological stage in radical prostatectomy patients. The potential role of the assay in predicting serum PSA recurrence after radical prostatectomy was explored. MATERIALS AND METHODS: We evaluated 100 radical prostatectomy candidates by reverse transcriptase polymerase chain reaction preoperatively, and status was compared to serum PSA, Gleason score and final pathological results. Potential surgical failure was defined as tumor at the surgical margin or extending into the seminal vesicle. Patients were monitored postoperatively by serum PSA every 4 months. Kaplan-Meier analysis was used to evaluate the correlation between reverse transcriptase polymerase chain reaction and disease recurrence, defined as a PSA of 0.2 ng/ml. or greater. RESULTS: Enhanced reverse transcriptase polymerase chain reaction for PSA had a stronger correlation with potential surgical failure than preoperative serum PSA or Gleason score (relative risks 15.2, 5.9 and 3.2, respectively). The correlation between these modalities and PSA recurrence was evaluated during a mean followup of 13.6 months (range 5 to 26). Of 36 patients with positive reverse transcriptase polymerase chain reactions 9 had failure by PSA compared to 3 of 64 (4.7%) with negative polymerase chain reactions (p<0.0286). The relative risk for failure by reverse transcriptase polymerase chain reaction was 3.6. Gleason score and serum PSA had higher correlations with postoperative PSA elevations (relative risk 13.2 and 7.6, respectively). A Cox regression analysis model demonstrated that reverse transcriptase polymerase chain reaction for PSA can be used in conjunction with Gleason score and provides statistically significant risk information. CONCLUSIONS: Enhanced reverse transcriptase polymerase chain reaction for PSA is a statistically significant predictor of potential failure by pathological analysis and of disease recurrence by PSA. Longer followup data are required to define further the role of the assay in the management of patients with prostate cancer.

The use of RT-PCR for prostate-specific antigen assay to predict potential surgical failures before radical prostatectomy: molecular staging of prostate cancer.

OBJECTIVE: To assess the potential role of a recently developed reverse transcriptase-polymerase chain reaction (RT-PCR) assay for prostate-specific antigen (PSA), that detects circulating prostate cells in patients with prostate cancer, in the management of clinically localized cancer. PATIENTS AND METHODS: A total of 138 men (mean age 62.5 years, range 49-70) scheduled for radical retropubic prostatectomy had an RT-PCR assay before surgery. The results were compared with the final pathological stage of disease, the results from local imaging techniques, serum PSA levels, digital rectal examination (DRE) and Gleason score. RESULTS: Enhanced RT-PCR for PSA was the best predictor of potential surgical failures; 70% of patients with positive surgical margins or invasion into the seminal vesicle were identified pre-operatively by a positive RT-PCR assay (odds ratio = 12.0, positive predictive value = 64%, negative predictive value = 87%). RT-PCR was able to identify pre-operatively patients with adverse pathology, despite low serum PSA values (< 4.0 ng/mL). In patients with high PSA level (> 10 ng/mL), RT-PCR discriminated between potentially curable candidates and those with established extraprostatic disease. CONCLUSIONS: RT-PCR for PSA adds unique prognostic information when considering patients for radical surgery. The final role for the RT-PCR assay is as yet undefined; however, the ability to detect potential surgical failures pre-operatively using a molecular approach should have a significant impact on the management of patients with prostate cancer.

Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo.

Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the bcl-2 protein. However, a recent immunohistochemical survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apoptosis-suppressing oncoprotein at significant levels (Colombel et al., Am. J. Pathol., 143: 390-400, 1993). Additionally, a number of hormone-refractory prostatic adenocarcinomas obtained from hormonally-treated patients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the parental or control-transfected cell lines, LNCaP/bcl-2 cells were highly resistant to a variety of apoptotic stimuli in vitro including serum starvation and 10 nM phorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay. s.c. injections of 10(6) LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of parental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2-transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli in vitro and suggest that such protection correlates with the ability to form hormone-refractory prostate tumors in vivo.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Molecular staging of prostate cancer. II. A comparison of the application of an enhanced reverse transcriptase polymerase chain reaction assay for prostate specific antigen versus prostate specific membrane antigen.

Current imaging modalities used to stage prostate cancer clinically fail to detect extracapsular disease in a significant subset of patients. A molecular based peripheral blood assay using the reverse transcriptase polymerase chain reaction has recently been shown to be a highly sensitive staging modality for detecting extraprostatic disease preoperatively. The assay uses primers that are specific for prostate specific antigen (PSA). We compare the application of the reverse transcriptase polymerase chain reaction assay using primers specific for the human prostate specific membrane antigen with results obtained from the same specimens by reverse transcriptase polymerase chain reaction for PSA. Prostate specific membrane antigen, a recently cloned prostatic antigen, is a transmembrane glycoprotein that has been described as prostate specific. These assays were applied to ribonucleic acids extracted from the peripheral blood lymphocyte fraction of 80 patients with clinically localized prostate cancer. In addition, blood specimens from 20 female patients, 20 young male patients, 25 age-matched control men under treatment for benign prostatic hypertrophy and 20 men with established, untreated metastatic prostate cancer were tested. All 3 groups of noncancer patients had negative polymerase chain reactions for PSA as well as prostate specific membrane antigen. Of 20 metastatic prostate cancer patients 16 (80%) had positive polymerase chain reactions for PSA, while only 10 (50%) had positive results for prostate specific membrane antigen. Among the 80 patients with clinically localized disease (stages T1 to T2cN0M0), 27 and 19 had positive polymerase chain reaction for PSA and prostate specific membrane antigen, respectively, from blood specimens obtained preoperatively. Analyzing the final pathology in each patient with the reverse transcriptase polymerase chain reaction assay identified a significantly stronger correlation with tumor invasion using the results of the PSA test rather than the results of the prostate specific membrane antigen reverse transcriptase polymerase chain reaction test (67% versus 34% sensitivity for detecting capsular penetration, 87% versus 46% sensitivity for detecting disease to the surgical margin and 83% versus 16% sensitivity for detecting seminal vesicle invasion). In contrast to the reverse transcriptase polymerase chain reaction assay for PSA, a similar assay done for prostate specific membrane antigen did not correlate with pathological stage of prostate cancer.

Enhanced reverse transcriptase-polymerase chain reaction for prostate specific antigen as an indicator of true pathologic stage in patients with prostate cancer.

BACKGROUND: As up to 50% of all patients with prostate cancer who have undergone radical prostatectomy are found to be understaged subsequent to surgery, a more sensitive early staging modality currently is needed. A molecular assay that detects prostate specific antigen (PSA)-synthesizing cells in the peripheral circulation of patients with prostate cancer is described. METHODS: An enhanced reverse-transcriptase polymerase chain reaction (RT-PCR) assay specific for PSA mRNA was performed on RNA extracted from blood drawn from 94 patients before radical prostatectomy. Surgical specimens were examined to determine the extent of tumor spread. The assay was compared with imaging modalities, digital rectal examination, and serum PSA level as predictors of pathology. Additionally, patients were monitored postoperatively by serum PSA level to determine any potential correlation between patient RT-PCR scores and subsequent tumor recurrence. RESULTS: Postoperative pathology revealed that 36 of the 94 patients had extraprostatic disease at the time of surgery. Enhanced RT-PCR identified 26 of these patients from preoperative blood specimens (72% sensitivity). The test was negative for 51 of the 58 patients with organ-confined disease (88% specificity). An odds ratio analysis showed that no other preoperative staging modality was related more strongly to extraprostatic or organ-confined disease. Follow-up PSA determinations revealed that RT-PCR positive patients were at higher risk for a recurrence. At 6 months after surgery, the rates for an increased PSA were 19 and 2% for RT-PCR-positive and -negative patients, respectively. CONCLUSIONS: The data from this follow-up study continue to support the utility of enhanced RT-PCR as an early staging modality for radical prostatectomy candidates.

Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay.

OBJECTIVE: Because up to 40 percent of surgically treated patients with prostate cancer are subsequently found to be clinically understaged, a more sensitive staging modality to identify extraprostatic disease prior to surgery is required. METHODS: We describe an enhanced reverse transcriptase [RT] polymerase chain reaction (PCR) assay utilizing oligonucleotide primers specific for the human prostate-specific antigen (PSA). This assay identifies PSA-synthesizing cells from reverse transcribed mRNA. This assay was applied to RNAs extracted from the peripheral blood lymphocytes of 65 patients with clinically localized prostate cancer. In addition, blood from 20 women, 20 young men, 25 age-matched control men under treatment for benign prostatic hyperplasia (BPH), and 18 men with established, untreated metastatic prostate cancer was tested. RESULTS: An RT-PCR assay for PSA can recognize one PSA-expressing cell diluted into one hundred thousand lymphocytes. The sensitivity of this assay can be enhanced by the addition of digoxigenin-modified nucleotides to the PCR reaction and this assay was applied to RNAs extracted from the peripheral lymphocyte fraction of 148 prostate cancer patients and controls at this institution. Although no specimen from women or men without cancer was positive in this assay, 14 of 18 metastatic prostate cancer patients were positive (77.8%). Additionally, 25 of 65 (38.5%) patients with clinically localized disease (T1-2b) were positive from blood specimens obtained prior to surgery. Final pathologic results from this group of patients identified a correlation between positivity on this assay and the presence of capsular tumor penetration (sensitivity, 68%; specificity, 84%) as well as strong correlation with the finding of carcinoma at the surgical margin (sensitivity, 87%; specificity, 76%). Logarithmic regression analysis of the results of the RT-PCR assay indicates its remarkable superiority to digital rectal examination, computed tomography scan, endorectal coil magnetic resonance imaging, PSA, prostate-specific antigen density, or Gleason score for predicting the true pathologic stage of prostate cancer in these surgically treated patients. CONCLUSIONS: An RT-PCR assay using PSA primers to detect prostate cells in the peripheral circulation of surgical-candidate patients is significantly correlated with capsular penetration and tumor-positive surgical margins. This molecular assay provides a sensitive and specific means to stage correctly apparent localized prostate cancer prior to radical prostatectomy.

Enhanced expression of p53 mRNA and protein in the regressing rat ventral prostate gland.

Previously, increased expression of mRNA encoding the p53 tumor suppressor protein was described during castration-induced regression of the rat ventral prostate gland with Northern blot techniques. This activity was confirmed with a ribonuclease protection assay that demonstrated a 16-fold induction of p53 transcripts in ventral prostate RNA within 72 hrs after castration. The induced expression of p53 mRNA correlated with increased detection of p53 protein in nuclei of regressing prostate epithelial cells. Immunohistochemical staining with anti-p53 antibody was strongly reactive for epithelial nuclei in castrated glands but unreactive for nuclei of control adult glands. In contrast to the upregulation of p53 in regressing prostate glands with a large proportion of apoptotic cells, expression of p53 mRNA was decreased in rat prostate glands that were stimulated to regrow by testosterone replacement.

Construction of tobacco mosaic virus subgenomic replicons that are replicated and spread systemically in tobacco plants.

Two tobacco mosaic virus (TMV)-derived replicons, created by deletion of most of the 126/183-kDa open reading frame (ORF), replicated and systemically invaded tobacco plants when supported by wild type TMV. One RNA replicon contained an internal direct repeat of 476 nucleotides from the 3' end of the 30-kDa ORF. Although this RNA was replicated, most of the progeny were heterogeneous in size and smaller than the original transcript. A second TMV-derived RNA replicon, without any internally repeated sequences and containing a deletion of the 5' portion of the 30-kDa ORF as well as most of the 126/183-kDa ORF, was created and coinoculated with wild type TMV as helper. This RNA also was replicated efficiently and systemically invaded tobacco plants. An examination of the sequences of cDNA clones obtained after PCR amplification of the progeny population of this RNA replicon demonstrated that the observed size heterogeneity was due to deletions and insertions adjacent to the artificially created deletion junction. These data demonstrate that a TMV infection is capable of supporting an artificially created RNA replicon, similar to defective interfering RNAs or satellites. However, these dependent RNAs were replicated without noticeably interfering with wild type TMV symptoms or replication.