FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice.

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions.

A phase I trial of pox PSA vaccines (PROSTVAC-VF) with B7-1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM) in patients with prostate cancer.

PURPOSE: Based on previous studies that demonstrated the safety profile and preliminary clinical activity of prostate specific antigen (PSA) targeted therapeutic vaccines, as well as recent laboratory data supporting the value of the addition of co-stimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) to these vaccines, we conducted a Phase I study to evaluate the safety and immunogenicity of a novel vaccinia and fowlpox vaccine incorporating the PSA gene sequence and TRICOM. METHODS: In this study, ten patients with androgen independent prostate cancer with or without metastatic disease were enrolled. Patients were treated with 2 x l0(8) pfu of a recombinant vaccinia virus vaccine (PROSTVAC-V) followed by 1 x 10(9) pfu of the booster recombinant fowlpox virus (PROSTVAC-F) both with gene sequences for PSA and TRICOM. The mean age of patients enrolled in the study was 70 (range 63 to 79). The mean PSA at baseline was 434 (range 9-1424). RESULTS: There were no deaths, and no Grade 3 or 4 adverse events. The most commonly reported adverse events, regardless of causality, were injection site reactions and fatigue. One serious adverse event (SAE) occurred that was unrelated to vaccine; this patient developed progressive disease with a new sphenoid metastasis. PSA was measured at week 4 and week 8. Four patients had stable disease (with less than 25% increase in PSA) through the week 8 study period. Anti-PSA antibodies were not induced with therapy: however, anti-vaccinia titers increased in all patients. CONCLUSION: This study demonstrated that vaccination with PROSTVAC-V and PROSTVAC-F combined with TRICOM is well-tolerated and generated an immune response to vaccinia. Therefore, PROSTVAC-VF/TRICOM represents a feasible therapeutic approach for further phase II and III study in patients with prostate cancer.

Retrospective stratification of a consecutive cohort of prostate cancer patients treated with a combined regimen of external-beam radiotherapy and brachytherapy.

PURPOSE: The evaluation of clinical variables that influence biochemical relapse-free survival in a cohort of patients treated by combined radiotherapy over a fixed interval. METHODS AND MATERIALS: Three hundred forty-eight patients diagnosed with clinical Stage T1--T3a prostate cancer were treated with a course of (103)Pd or (125)I brachytherapy followed by a limited course of external beam radiation formed the basis for study. All censored patients had a minimum 2-year follow-up. Biochemical relapse-free survival (BRFS) was estimated using a modified American Society for Therapeutic Radiology and Oncology consensus definition. Discrete "risk groups" were developed based on BRFS as influenced by pretreatment parameters. RESULTS: Significant risk factors contributing to biochemical failure were serum prostate-specific antigen (PSA) greater than 20 ng/mL, Gleason sum of 7 or greater, or clinical stage T2c or greater. Five-year biochemical control for those exhibiting no risk factor was 88%; one risk factor, 75%; two or more risk factors, 51%. The differences in BRFS among all three risk groups were statistically significant. Outcomes for patients presenting with PSA 10 to 20 ng/mL, but otherwise low-risk disease, fared no differently from those low risk patients presenting with PSA less than 10 ng/mL. CONCLUSIONS: Combined radiotherapy with (103)Pd or (125)I followed by external beam radiotherapy achieves a high rate of biochemical and clinical control in patients with low- to intermediate-risk clinically organ confined disease.

Outbreak of bloodstream infections associated with dialysis machine waste ports in a hemodialysis facility.

Eight cases of gram-negative bloodstream infection without a clinically evident source occurred at a hemodialysis unit in Jerusalem between February and September 1997. All infections could be traced to three of the 13 dialysis machines in use. Epidemiological investigation, including pulsed-field gel electrophoretic characterization of organisms isolated from the patients and dialysis machines, implicated the Waste Handling Option system of the machines as the source of the infections. Discontinuation of the Waste Handling Option system use was associated with prompt cessation of the outbreak.

Infected nonunion of the tibia.

The treatment of infected nonunited fractures of the tibia using the techniques of Ilizarov was compared with autogenous cancellous bone graft application under a well vascularized soft tissue envelope. There were 10 patients in the Ilizarov group and 17 in the bone graft group. Soft tissue coverage with a free vascularized or a rotational muscle flap was used more frequently among the patients having bone graft (71%) than the Ilizarov group (30%). All 27 patients had bony defects (average, 3.7 cm; range, 1-18 cm). At an average followup of 6 years, 26 patients had a functional limb, and one patient (Ilizarov group) ultimately required a below knee amputation. Three patients in each group required a second plate and bone graft procedure to gain union. Infection persisted in four patients (all in the Ilizarov group). If a well vascularized soft tissue envelope is present (particularly after flap coverage), bone grafting procedures are safe and efficacious. The Ilizarov technique may be best suited for the treatment of very proximal or distal metaphyseal nonunions and nonunions associated with large leg length discrepancies.

Effect of prostate biopsy on the results of the PSA RT-PCR test.

OBJECTIVES: To evaluate the effect of prostate biopsy on the prostate-specific antigen (PSA) reverse transcriptase-polymerase chain reaction (RT-PCR) test. METHODS: Ninety men who were scheduled to undergo prostate biopsy because of an elevated PSA or abnormal digital rectal examination, or both, were recruited to have PSA RT-PCR performed on peripheral blood samples drawn before and at 30 minutes, 1 week, and 1 month after undergoing prostate biopsy. PSA RT-PCR was performed and all PCR products were blotted and hybridized with phosphorus-32 (32-P)-PSA cDNA probe (exon 3 to 5). RESULTS: Of 90 patients, 77 had a negative prebiopsy PSA RT-PCR result. Of these 77, 2 (2.6%) had a positive PSA RT-PCR result at some point after the biopsy procedure. Both of these patients had no evidence of malignancy on biopsy. The PSA RT-PCR test was positive at 30 minutes for 1 patient, but was negative at 1 week; the other was positive at 1 week but the patient did not return for the 1-month sample. CONCLUSIONS: Our study indicates that 2.6% of patients with an initially negative PSA RT-PCR will have a positive PSA RT-PCR test after biopsy has been performed. Although this is uncommon, it may have profound implications for those patients in whom it occurs. On the basis of our results, it appears that one should wait longer than 1 week after prostate biopsy before obtaining blood for PSA RT-PCR testing to decrease the likelihood of a spurious PSA RT-PCR result.

Prostate-specific membrane antigen.

BACKGROUND: In an effort to discover new prostate-specific antigens (PSAs) to enhance our understanding of the functions and behavior of the prostate and the complex processes involved in prostate tumor progression, the structure and function of the PSM antigen has been elucidated. METHODS: The PSM antigen was recognized using the 7E11-C5.3 monoclonal antibody, generated against the LNCaP human prostate adenocarcinoma cell line. The PSM cDNA was isolated by PCR, using tryptic peptides of immunoprecipitated PSM to design degenerate primers. RESULTS: The prostate specific membrane antigen (PSM) is a 100 KD glycoprotein which appears to be a type II integral membrane protein. The protein and cDNA have been extensively characterized and the findings reviewed in the report. CONCLUSIONS: PSM, a new prostate antigen is valuable as a marker for hematogenous micro-metastatic tumor dissemination as detected in RT-PCR assays of peripheral blood. PSM has many properties that may be potentially useful as a molecular target in monoclonal antibody directed strategies of tumor imaging and therapy.

Prostate-specific membrane antigen and other prostatic tumor markers on the horizon.

The PSM antigen is an exciting new molecule with many potentially valuable applications. Further research with PSM may help us to elucidate the complex process of prostatic neoplasia better. Current avenues of research with PSM include the generation of new and improved monoclonal antibodies targeting different portions of PSM and PSM', which may improve the results of imaging and targeting prostate cancer. Gene therapy using the PSM promoter to drive prostate-specific expression of various cytokines and other factors is another exciting potential application deserving of attention, and refinement of serum PSM assays may greatly add to the present array of diagnostic modalities offered to patients with suspected prostate cancer. Thus, PSM is a potentially valuable addition to our armamentarium of prostate markers. Additionally, a host of other potential markers to increase our understanding of the complex biology of the normal and malignant prostate are on the horizon. Just how far away that horizon is awaits further basic and clinical investigations.

Can prostate-specific antigen reverse transcriptase-polymerase chain reaction be used as a prospective test to diagnose prostate cancer?

The present study addressed the question as to whether prostate-specific antigen reverse transcriptase-polymerase chain reaction (PSA RT-PCR) could be used to identify prospectively men who have prostate cancer and to help determine which patients with an initially negative biopsy would benefit from rebiopsy. PSA RT-PCR was performed prospectively on 90 patients who were to have a prostate biopsy because of an elevated PSA level, an abnormal digital rectal examination, or both. PSA RT-PCR was performed, and the sensitivity of the test was enhanced by hybridization of the PCR with a 32P-labeled PSA cDNA probe (exons 3-5). Of the 90 men, 36 (40%) had prostate cancer on biopsy. Of these 36 men, 5 (13.9%) had a positive PSA RT-PCR finding, whereas 31 (84.1%) tested negative. Of 54 men with negative biopsies, 8 (14.8%) had a positive PSA RT-PCR result. The sensitivity of PSA RT-PCR for the detection of biopsy-proven prostate cancer was 13.9% and the specificity was 85.2%. Only 3 of 12 (25%) patients with advanced disease had a positive test result. The sensitivity of PSA RT-PCR for the detection of biopsy-proven prostate adenocarcinoma in men suspected of having prostate cancer is poor. Indeed, men without biopsy-proven prostate cancer are just as likely to have a positive result in the PSA RT-PCR as are men with cancer. Whether these men with negative prostate biopsies and positive PSA RT-PCR findings may eventually develop prostate cancer remains to be determined. At this time, PSA RT-PCR for the prospective detection of prostate cancer should be considered investigational.

Bilateral renal oncocytomatosis in a patient with renal failure.

Bilateral multifocal renal oncocytomas are very rare disorders with only 6 previously reported cases in the world literature, of which only 3 have had pathologic confirmation. We present the first reported case of diffuse, bilateral, multifocal renal oncocytomatosis in a patient with end-stage renal disease requiring hemodialysis. Our patient was found to have hundreds of nodular tumors in both kidneys on exploration, representing the second such reported finding.

Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11.

The prostate-specific membrane antigen (PSM) was identified by the monoclonal antibody 7E11-C5.3, which was raised against the human prostatic carcinoma cell line LNCaP (3). The PSM antigen is expressed by normal, neoplastic, and metastatic prostatic tissues. The 2.65-kb cDNA encoding the 100-kDa PSM glycoprotein was cloned from LNCaP cells (4). Studies have shown that the expression of PSM is tissue-specific (5). In the present study monochromosomal somatic cell hybrids were used to localize the PSM gene to human chromosome 11. Using this information, initial mapping studies identified two potential PSM gene loci at 11p11.1-p13 and 11q14. Further high-stringency analysis using cosmid probes identified the 11q14 region as the location of the PSM gene.

Sensitive detection of prostatic hematogenous tumor cell dissemination using prostate specific antigen and prostate specific membrane-derived primers in the polymerase chain reaction.

We developed a polymerase chain reaction based assay enabling sensitive detection of hematogenous tumor cell dissemination in patients with prostate cancer. We performed "nested polymerase chain reaction," amplifying messenger ribonucleic acid sequences unique to prostate specific antigen (PSA) and to the prostate specific membrane antigen, and compared the respective results. Prostatic tumor cells were detected in 2 of 30 patients (6.7%) by polymerase chain reaction with PSA derived primers, while prostate specific membrane primers detected tumor cells in 19 (63.3%). All 16 negative controls had negative PSA and prostate specific membrane polymerase chain reaction. Assays were repeated to confirm results, and polymerase chain reaction products were verified by deoxyribonucleic acid sequencing and Southern analysis. Patients harboring circulating prostatic tumor cells as detected by prostate specific membrane and not by PSA polymerase chain reaction included 7 of 13 previously treated by radical prostatectomy who had nonmeasurable serum PSA levels at the time of this assay. The significance of these findings with respect to future disease recurrence and progression will be investigated.

Reversal by transferrin of growth-inhibitory effect of suramin on hormone-refractory human prostate cancer cells.

BACKGROUND: The second leading cause of cancer-related deaths in American men is metastatic hormone-refractory adenocarcinoma of the prostate, for which there is currently no effective treatment. Transferrin is abundant in bone stroma and has been found to stimulate models of hormone-refractory metastatic prostate cancer. Suramin, a compound that has been used to treat metastatic prostate cancer, has been demonstrated to antagonize the binding of transferrin to the transferrin receptor and to suppress uptake of iron by hematopoietic cells. PURPOSE: The purpose of our study was to determine whether transferrin may reverse the inhibitory action of suramin on metastatic prostate-derived cell lines. METHODS: Five human prostate cell lines (PC-3, PC-3M, DU-145, TSU-Pr1, and LNCaP) derived from metastatic deposits were examined for response to growth stimulation by apotransferrin, for the presence of transferrin receptors by binding of 125I-labeled transferrin, and for relative transferrin receptor messenger RNA (mRNA) content by ribonuclease protection assays. We measured the amount of growth inhibition by suramin in low serum assays to demonstrate maximal inhibition over the apotransferrin to reverse the inhibition of suramin in these tumors. RESULTS: The results clearly demonstrate that the androgen-insensitive metastatic cell lines (PC-3, PC-3M, DU-145, and TSU-Pr1) demonstrate increased cell numbers when exposed to holotransferrin or apotransferrin, while the androgen-sensitive cell line (LNCaP) did not show any increase. All cell lines demonstrated a similar number of transferrin receptors and transferrin receptor mRNA. We used these maximally inhibitory, but clinically relevant, concentrations of suramin to determine whether transferrin could reverse the inhibition, and it did, but only in the androgen-insensitive metastatic lines. Indeed, in the PC-3 cells, inhibition turned to stimulation with the addition of transferrin, and even at the highest concentration of suramin tested, 400 microM, a concentration that would be toxic to patients, the amount of inhibition by suramin was still reduced by more than 50% by transferrin in TSU-Pr1 cells. In the androgen-sensitive LNCaP cells, however, transferrin had limited ability to block the inhibitory activity of suramin. CONCLUSIONS: Concentrations of tumor-stimulating factors, such as transferrin, in the metastatic microenvironment need to be taken into consideration in the use of suramin and suramin-like derivatives. Novel strategies need to be identified that will negate the action of transferrin on androgen-insensitive cells.

Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate-specific membrane antigen and prostate-specific antigen-based assays.

A highly sensitive nested reverse transcriptase-PCR assay, with primers derived from the prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) cDNA sequences, has been used to detect occult hematogenous micrometastatic prostate cells. In 77 patients with prostate cancer, PSM and PSA primers detected circulating prostate cells in 48 (62.3%) and 7 (9.1%) patients, respectively. In treated stage D disease patients, PSM primers detected cells in 16 of 24 patients (66.7%), while PSA primers detected cells in 6 of 24 (25%). In post-radical prostectomy patients with negative serum PSA values, PSM primers detected metastases in 21 of 31 patients (67.7%), whereas PSA primers detected cells in only 1 of 33 (3.0%), indicating that micrometastatic spread may be a relatively early event in prostate cancer. The analysis of 40 individuals without known prostate cancer provides evidence that this assay is highly specific and suggests that PSM expression may predict the development of cancer in patients without clinically apparent prostate cancer. Using PSM primers, we detected micrometastases in 4 of 40 controls, 2 of whom had known benign prostatic hyperplasia and were later found to have previously undetected prostate cancer. The clinical significance of detection of hematogenous micrometastic prostate cells using PSM primers and potential applications of this molecular assay, as well as the assay for PSA, merit further study.

Expression of the prostate-specific membrane antigen.

We have recently cloned a 2.65-kilobase complementary DNA (cDNA) encoding the prostate-specific membrane antigen (PSM) recognized by the 7E11-C5.3 anti-prostate monoclonal antibody. Immunohistochemical analysis of the LNCaP, DU-145, and PC-3 prostate cancer cell lines for PSM expression using the 7E11-C5.3 antibody reveals intense staining in the LNCaP cells with no detectable expression in both the DU-145 and PC-3 cells. Coupled in vitro transcription/translation of the 2.65-kilobase full-length PSM cDNA yields an M(r) 84,000 protein corresponding to the predicted polypeptide molecular weight of PSM. Posttranslational modification of this protein with pancreatic canine microsomes yields the expected M(r) 100,000 PSM antigen. Following transfection of PC-3 cells with the full-length PSM cDNA in a eukaryotic expression vector, we detect expression of the PSM glycoprotein by Western analysis using the 7E11-C5.3 monoclonal antibody. Ribonuclease protection analysis demonstrates that the expression of PSM mRNA is almost entirely prostate specific in human tissues. PSM expression appears to be highest in hormone-deprived states and is hormonally modulated by steroids, with 5-alpha-dihydrotestosterone down-regulating PSM expression in the human prostate cancer cell line LNCaP by 8-10-fold, testosterone down-regulating PSM by 3-4-fold, and corticosteroids showing no significant effect. Normal and malignant prostatic tissues consistently show high PSM expression, whereas we have noted heterogeneous, and at times absent, expression of PSM in benign prostatic hyperplasia. LNCaP tumors implanted and grown both orthotopically and s.c. in nude mice abundantly express PSM, providing an excellent in vivo model system to study the regulation and modulation of PSM expression.

Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen.

Recently, a novel M(r) 100,000 prostate-specific membrane glycoprotein (PSM) has been detected by the prostate-specific monoclonal antibody 7E11-C5, raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed exclusively by normal and neoplastic prostate cells and metastases. We now report the molecular cloning of a full-length 2.65-kilobase complementary DNA encoding the PSM antigen from a human LNCaP complementary DNA library by polymerase chain reaction using degenerate oligonucleotide primers. Analysis of the complementary DNA sequence has revealed that a portion of the coding region, from nucleotide 1250 to 1700, has 54% homology to the human transferrin receptor mRNA. The deduced polypeptide has a putative transmembrane domain enabling the delineation of intra- and extracellular portions of this antigen. In contrast to prostate-specific antigen and prostatic acid phosphatase which are secreted proteins, PSM as an integral membrane protein may prove to be effective as a target for imaging and cytotoxic targeting modalities.

Upstream regulatory elements of murine alpha 4-interferon gene confer inducibility and cell type-restricted expression.

We have identified and functionally characterized DNA sequences that are required for the inducible and cell-restricted expression of the murine alpha 4-interferon gene. Hybrid plasmids in which the alpha 4 promoter region or its 5' deletions were inserted upstream of the CAT gene were constructed, and the expression of these hybrid genes was studied in mouse L-cells both in permanent and transient assays with comparable results. Inducible expression was not affected by deletions up to -109; however, when the deletion was extended to -96, inducibility by Newcastle disease virus was abolished; however, this hybrid plasmid was expressed constitutively. Further deletion to -88 did not permit either constitutive or inducible expression. Insertion of the 35-base pair-long sequence (-109 to -75 base pairs) from the alpha 4 promoter region 5' of the minimal alpha 4 or human immunodeficiency virus promoter region, conferred inducibility to these two inactive promoters. The 5' deleted hybrids or plasmids containing the inducible element were induced only at low levels in transfected NIH/3T3 cells that do not express endogenous alpha 4 gene efficiently, indicating that the inducible region also determines the cell-specific expression. A tandem repeat of AGTGAA, which is present in the -109 to -88 region of alpha 4 in two copies, showed both basal levels of expression and inducibility in L-cells, while its analogue AATGAA was highly inducible but was not expressed constitutively. The inducibility of the synthetic hexamer repeats did not show cell type-restricted expression, suggesting that their response does not fully reflect the range of expression observed for the inducible region and the endogenous alpha genes.

Synthesis, antiviral activity, and conformational characterization of mouse-human alpha-interferon hybrids.

Reciprocal hybrids were constructed between human and mouse interferons (IFNs), and their antiviral activity was examined on different target cells and compared to the activity of the parental molecules. In addition, we used a number of predictive algorithms on a data base of the available alpha-interferon sequences to propose a working model for the overall conformation of the alpha-interferon molecule that is consistent with the structural predictions. Remarkable conservation within the predicted alpha-helical segments of the interferon molecule was observed. We propose that the observed changes in the activity and specificity of the hybrids obtained are largely due to the sequences present in the loops at the ends of the major helical structures; these are less conserved, contain beta-bends, and are generally hydrophilic and flexible. The data on the constructed mouse-human hybrids have shown that the activity on human cells is contributed by determinants present in the N-terminal 122 amino acids of human IFN, thus implicating one or more loops within this region (e.g. loops 1-12, 25-38, 70-74, and 103-113). The activity on bovine cells appears to be localized mainly in sequence 60-121, implicating the role of loops 70-74 and/or 103-113 of the human IFN molecule. The specificity of mouse IFN for mouse cells is in some or all of the loops (70-74, 103-113, 134-139, and 163-166) in the C-terminal sequence. The proposed working model should provide guidelines for the study of the specificity of action in molecular terms.

Pulmonary function screening and monitoring in occupational health.

Lung function testing among occupationally exposed workers has demonstrated its usefulness in risk evaluation and, once risks are identified, in medical surveillance. Its usefulness for screening and biological monitoring for effects of exposure is not yet established; in part, this is due to some failure to understand the requirements for screening and for monitoring, and in part, it is due to some unresolved questions as to procedures and their interpretation. The use of screening in occupational groups in general and those exposed to inhalational risks is evaluated. The current recommendation is that priority for screening be given to risk factor reduction, including smoking cessation. Problems associated with screening relate to procedures, equipment, and interpretation of data. An agenda is proposed for an international effort to determine principles for epidemiological use of lung function tests for prevention of occupational pulmonary disease risks.