B. Jill Williams

SUPPRESSION OF PRIMARY TUMOR GROWTH AND THE PROGRESSION TO METASTASIS WITH p53 ADENOVIRUS IN HUMAN PROSTATE CANCER

PURPOSE Numerous advances have been made in gene therapy approaches for the treatment of solid tumors, including prostate cancer. While treatment of the primary tumor has been well investigated, little information is available regarding gene therapy techniques which might impact on the progression to metastatic disease. We investigate the ability of p53 adenovirus to suppress not only primary tumor growth, but also the progression to metastatic disease. Mutation of the p53 tumor suppressor gene has been associated with the progression of prostate cancer. In this study, we utilized a metastatic model for human prostate cancer to determine if introduction of the wild-type p53 gene using an adenoviral vector (rAd-p53) impacted on primary tumor growth as well as the progression to metastatic disease. MATERIALS AND METHODS For our studies, we used the human prostate cancer cell line PC-3, which has a homozygous loss of p53 expression. Expression of exogenous p53 as well as p21 induction at various time points after infection with rAd-p53 was determined in vitro. In vivo studies were performed in nude mice following orthotopic (intraprostatic) injection of PC-3 cells. Primary tumor growth as well as the progression to metastatic disease was assessed following rAd-p53 treatment. RESULTS In vitro studies demonstrated high levels of p53 gene expression as well as the induction of p21 gene expression. Infection of PC-3 cells with rAd-p53 resulted in marked growth inhibition, as well as wide-spread fragmentation of nuclei and secretion of nuclear matrix proteins into the culture medium consistent with the process of apoptosis. In vivo studies demonstrated that a single injection of rAd-p53 into an established orthotopic prostate tumor resulted not only in primary tumor growth suppression (treated = 97.5 +/- 25.3 mm.3 versus control = 393.4 +/- 67.2 mm.3; p = 0.0002) but also reduced the frequency of progression to metastatic disease (treated = 8 of 19 versus control = 18 of 19; p = 0.001). CONCLUSION These experiments demonstrate that a single injection of rAd-p53 into an established orthotopic prostate tumor results not only in suppression of primary tumor growth, but also in a reduction of the frequency of progression to metastatic disease. These results suggest that a rAd-p53 gene therapy strategy may be useful in the treatment of human prostate cancer.

Serum testosterone levels in african-american and white men undergoing prostate biopsy

OBJECTIVES Because androgen levels are known to influence prostate growth, we performed a prospective analysis of serum testosterone levels in all African-American and white men who underwent transrectal ultrasound-guided prostate biopsies to evaluate an abnormal digital rectal examination (DRE) and/or serum prostate-specific antigen (PSA) level greater than 4 ng/mL. METHODS From June 1996 through July 1998, we evaluated 453 men (189 African-American and 264 white men) who underwent prostate needle biopsy because of an abnormal DRE or serum PSA greater than 4 ng/mL, or both. All men had morning serum testosterone levels determined just before undergoing prostate needle biopsy. Serum testosterone levels were compared on the basis of the prostate biopsy result (positive or negative for prostate cancer) and by race. RESULTS A total of 453 men underwent prostate biopsy and had morning serum testosterone levels available for comparison. Of the 264 white men who underwent biopsy, 88 (33%) were found to have prostate cancer compared with 67 (35%) of 189 African-American men who underwent biopsy. In the white men without cancer, the mean serum testosterone level was 380. 19 ng/dL; those with prostate cancer had a mean serum testosterone level of 419.52 ng/dL. The mean serum testosterone level in African-American men without cancer was 424.30 ng/dL; it was 386.55 ng/dL in those with prostate cancer. There was no statistical difference in serum testosterone levels based on biopsy result or race. CONCLUSIONS Although several studies have suggested that African-American men have higher serum testosterone levels than white men, these differences were noted only in men 40 years of age or younger. As was noted in our study, after age 40, African-American and white men have comparable serum testosterone levels. In addition, although prostate growth is androgen dependent, we found no difference in serum testosterone levels in men with and without prostate cancer.

Amphiregulin Is Coordinately Expressed with Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Interstitial Smooth Muscle of the Human Prostate

Peptide growth factors have been proposed as mediators of smooth muscle-epithelial cell interactions in the human prostate; however, the identity of these molecules has not been established. In this study, we compared expression levels of messenger RNAs (mRNAs) encoding the epidermal growth factor (EGF) receptor-related receptor tyrosine kinases (ErbB1 through 4), the six EGF receptor ligands, EGF, transforming growth factor (TGF)-a, amphiregulin (ARG), HB-EGF, betacellulin, and epiregulin, and the related molecule heregulin-a ,i n a series of 10 prostate tissue specimens. Only EGF showed a diseasespecific association, with increased mRNA levels in four of five PCa specimens in comparison to matched normal tissue from the same subject. In contrast, ARG and HB-EGF mRNAs showed a coordinate pattern of expression in 7/10 specimens that was distinct from all other growth factor or receptor genes examined and from mRNAs for prostate specific antigen, the androgen receptor and GAPDH, a housekeeping enzyme. Analysis of an additional series of benign prostatic hyperplasia and prostate cancer specimens from 60 individuals confirmed that ARG and HB-EGF mRNA levels varied in a highly coordinate manner (r 5 0.93; P , 0.0001) but showed no association with disease. ARG was immunolocalized largely to interstitial smooth muscle cells (SMC), previously identified as the site of synthesis of HB-EGF in the prostate, while the cognate ARG and HB-EGF receptor, ErbB1, was localized exclusively to ductal epithelial cells and carcinoma cells. Although ARG was a relatively poor mitogen for Balb/c3T3 cells in comparison to HB-EGF, it was similar in potency to HB-EGF in stimulating human prostate epithelial cell growth, suggesting that prostate epithelia may be a physiologic target for ARG in vivo. Expression of both ARG and HB-EGF mRNAs was induced in cultured prostate SMC by fibroblast growth factor-2, a human prostate SMC mitogen linked to prostate disease. These findings indicate that ARG and HB-EGF are likely to be key mediators of directional signaling between SMC and epithelial cells in the human prostate and appear to be coordinately regulated. (Endocrinology 140: 5866 ‐5871, 1999)

RUNX1 (AML-1) and RUNX2 (AML-3) cooperate with prostate-derived Ets factor to activate transcription from the PSA upstream regulatory region

The RUNX transcription factors (RUNX1, RUNX2, and RUNX3) play essential roles in hematopoiesis and skeletal development. Consistent with these roles in differentiation and cell cycle, the activity of both RUNX1 and RUNX3 is perturbed in cancer. To determine a role for the RUNX factors in prostate biology, we investigated the expression of RUNX factors in prostate epithelial cell lines and normal prostate tissue. RUNX1, RUNX2, and RUNX3 were expressed in both normal prostate tissue and an immortalized, non‐transformed cell line. We found that prostate cancer‐derived cell lines expressed RUNX1 and RUNX2, but not RUNX3. Next, we sought to identify prostate‐specific genes whose expression could be regulated by RUNX proteins. Four consensus RUNX sites are located within the prostate‐specific antigen (PSA) regulatory region. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 is specifically bound to the PSA regulatory region in LNCaP cells. RUNX1 and RUNX2 activated the PSA regulatory region alone or cooperatively with prostate‐derived ETS factor (PDEF) and RUNX1 physically associated with PDEF. Taken together, our results suggest that RUNX factors participate in prostate epithelial cell function and cooperate with an Ets transcription factor to regulate PSA gene expression. J. Cell. Biochem.

Detection of eIF4E gene amplification in breast cancer by competitive PCR.

AbstractBackground: Initiation factor eIF4E binds to mRNA as the initial step for protein translation. Overexpression of the eIF4E oncoprotein has been found in breast cancer but not in benign breast tissue. The objective of this study is to determine if eIF4E oncoprotein overexpression is associated with eIF4E gene amplification in breast cancer using Western blots and competitive polymerase chain reaction (PCR). Methods: Unknown concentrations of DNA extracted from breast specimens were amplified by PCR using a set of primers spanning intron 2/exon 3 of the eIF4E gene. In the same PCR tube, an internal control consisting of a known concentration of an eIF4E DNA template with 20-base pair (bp) deletion was used as the competitive reference standard (CRS) for competitive PCR. Gel electrophoresis of the PCR products was performed and the bands quantified by densitometry. eIF4E gene amplification was then determined relative to a nonamplified gene (gastrin). Using an anti-eIF4E rabbit antibody, Western blots were performed on benign and malignant breast specimens. Quantification was accomplished by developing blots with a color assay using nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), scanned and analyzed by densitometry. Results: Twenty-two breast specimens (14 cancer, 8 control) from patients were examined for eIF4E gene amplification and oncoprotein expression. In all fourteen specimens from stage I–III breast cancer patients, eIF4E overexpression was detected at 3- to 30-fold (16.71±7.83) elevations. Similarly, all 14 specimens demonstrated eIF4E gene amplification by competitive PCR (3.69±1.27). In the eight benign breast specimens examined, all were negative for eIF4E overexpression and gene amplification. Conclusions: Overexpression of eIF4E was associated with eIF4E gene amplification in breast cancer specimens. No overexpression or gene amplification was detected in benign breast tissues. eIF4E gene amplification may be one mechanism for eIF4E oncoprotein overexpression.

Cancer-specific targeting of an adenovirus-delivered herpes simplex virus thymidine kinase suicide gene using translational control.

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV‐1 thymidine kinase (HSV‐Tk), was modified with a complex 5′ upstream‐untranslated region (5′‐UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor‐specific gene delivery was optimized by incorporation of the 5′‐UTR‐modified suicide gene (HSV‐UTk) into an adenovirus vector (Ad‐CMV‐UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF‐7, MDA‐MB435, and ZR‐75‐1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) was used to quantify HSV‐Tk mRNA for cells infected with Ad‐CMV‐UTk as well as with Ad‐CMV‐Tk (a control adenovirus in which HSV‐Tk is not regulated at the level of translation). Translation of HSV‐Tk in the Ad‐infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro‐drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell‐specific expression of HSV‐Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad‐CMV‐Tk or Ad‐CMV‐UTk. These data collectively showed enhanced cancer cell‐specific gene expression and reduced normal tissue gene expression for the Ad‐HSV‐UTk compared to the Ad‐CMV‐Tk, leading to increased cancer cell‐enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity. Copyright

Tensile strength of cadaveric fascia lata compared to small intestinal submucosa using suture pull through analysis.

PURPOSE The modified pubovaginal sling has become popular as first line treatment for stress urinary incontinence. With the increasing use of cadaveric fascia as a sling material, widespread shortages are prevalent, hence limiting its availability. The increased morbidity with the use of synthetic sling materials and autologous fascia has stimulated investigation of other sling materials. We evaluated the tensile strength of 4 suture types, and compared tensile strength of cadaveric fascia lata to porcine small intestinal submucosa using suture pull through analysis to assess their efficacy and durability for use in anti-incontinence procedures. MATERIALS AND METHODS Suture breaking load was determined using 2 and 1-zero polypropylene suture, and 2 and 1-zero polyglactin suture. Freeze dried gamma irradiated human fascia lata and freeze-dried small intestinal submucosa were evaluated. Suture was fixed to sling material using the cross fold technique. Mean suture breakage and suture pull through were determined using a tensionometer by measuring the load applied to the sling/suture system. Statistical analysis was performed. RESULTS Mean suture breakage load was greatest with 1-zero polyglactin (8.10 pounds) and least with 2-zero polypropylene (3.68 pounds). Mean suture breakage strength was similar for 1-zero polypropylene and 2-zero polyglactin at 5.26 and 5.40 pounds, respectively. Mean suture pull through load using 1-zero polypropylene suture and the cross fold technique was 5.64 pounds for cadaveric fascia and 2.74 pounds for small intestinal submucosa (p <0.0001). Maximum load was limited by the suture strength when using cadaveric fascia, whereas, maximum load was limited in small intestinal submucosa by its inherent tensile strength. However, using a new technique for suture fixation to the small intestinal submucosa, we were able to increase significantly mean suture pull through load to 3.36 pounds (p = 0.008). Additionally, with this new technique small intestinal submucosa allowed gross stretching before suture pull through that was not seen with cadaveric fascia. CONCLUSIONS Despite the current standard use of 1-zero polypropylene suture for pubovaginal sling fixation, our data suggest that 1-zero polyglactin suture is the strongest, and its use with pubovaginal sling fixation warrants further investigation. Using the cross fold technique and 1-zero polypropylene suture, tensile strength was greatest with cadaveric fascia compared to small intestinal submucosa. Although small intestinal submucosa was not as strong as cadaveric fascia, our persuasive preliminary data suggest that further investigation is warranted in the use of small intestinal submucosa and other suture fixation techniques, and its observed stretch capacity. Hence, with further studies small intestinal submucosa may remain a viable option for pubovaginal sling material.

Bovine dermis: a novel biologic substitute for autologous tissue in sling surgery

Women at high risk for sling failure (advanced age, previous surgical failure, and intrinsic sphincter deficiency) underwent either bovine dermis (BOV) or autologous rectus fascia (ARF) pubovaginal sling at two hospitals. “Global cure” encompassed stress, emptying, anatomic, protection, and instability (SEAPI) composite = 0 and subjective satisfaction. Cure of stress urinary incontinence (SUI cure) equaled SEAPI (S) = 0 and negative cough-stress test. Eighty-five women (48 ARF, 37 BOV) completed a 12-month evaluation. Preoperative SEAPI and quality of life indices (QOL) were not statistically different (NS). “Global cure” for ARF and BOV was 60.4% and 54.1%, respectively (NS). “SUI cure” for ARF and BOV was 81.3% and 83.8%, respectively (NS). Postoperative SEAPI and QOL indices were significantly improved for each material; improvement was similar between ARF and BOV groups (NS). BOV is a promising substitute for ARF in women at high risk for surgical failure. Longer follow-up is needed before indications for this material are expanded.

DETERMINANTS OF FAILURE AFTER SLING SURGERY FOR FEMALE STRESS URINARY INCONTINENCE

Results: Of 728 women, 177 (24.3%) underwent autologous rectus fascia bladder neck slings, 259 (35.6%) underwent porcine dermis bladder neck slings, and 292 (40.1%) underwent polypropylene midurethral slings. The mean follow-up period for the entire cohort was 42 months. The “SUI cure” rate was 75.4% and “global cure” rate was 54.9% for the entire cohort. Of the 153 women (21%) who achieved SUI cure but not global cure, 143 (93.5%) had a SEAPI subjective composite score > 0. The reasons for failure included: emptying (43.1%), anatomy or bladder neck descent (9.2%), pad use (20.3%), and inhibition / urge incontinence (66%). A VAS < 8 was recorded in 36 (23.5%) women and 10 of these women (6.5%) failed for reasons not included in SEAPI (e.g. posterior and apical prolapse, pelvic and abdominal pain, dyspareunia, and urgency without incontinence). Despite women undergoing 3 different sling procedures differing in some demographic, urodynamic, and perioperative criteria, reasons for failure were not statistically different between sling types. A statistically significant improvement in all QOL indices was seen in “SUI cure” and failure groups, as well as “global cure” and failure groups. Interpretation of results: Although the populations of women undergoing surgery with different sling materials differed from one another, the rate of SUI resolution appears to be similar. Likewise, the incidence of postoperative urinary urge incontinence, voiding dysfunction, pad use, and bladder neck mobility are also similar. If these factors are considered, along with symptoms not accounted for by the SEAPI scale, then the true cure rate after sling surgery is more modest than originally quoted. Despite the different cure rates, women appear to have a significant improvement in their quality of life after sling surgery.

IS SLING STIFFNESS ASSOCIATED WITH POSTOPERATIVE VOIDING DYSFUNCTION? A COMPARISION OF TWO POLYPROPYLENE MIDURETHRAL SLINGS

INTRODUCTION AND OBJECTIVES: Recent modifications to midurethral slings (MUS) by some manufacturers have resulted in stiffer slings which are less resistant to deformation in vitro. The performance of these materials after implantation and the incidence of voiding dysfunction have not been fully investigated. We retrospectively report on the postoperative voiding dysfunction after two types of suprapubic (SP) and transobturator (TO) MUS: high-stiffness (Bard) and low-stiffness (AMS). METHODS: Group 1 consisted of 80 consecutive women who underwent high-stiffness MUS (40 TO, 40 SP), under a manufacturerinstitutional agreement. Group 2 consisted of the most recent 80 consecutive women who underwent an established low-stiffness MUS (40 TO, 40 SP). All procedures were performed by one surgeon using standard placement and tensioning technique. All voiding trials were performed per protocol. Postoperative voiding dysfunction was subcategorized by: asymptomatic high post void residual (PVR), positional voiding, urinary retention < 30 days, and retention requiring urethrolysis. Secondary outcomes included validated quality of life (QOL) questionnaires. RESULTS: Of 40 women undergoing high-stiffness SP sling, 17 (42.5%) women had voiding dysfunction: high PVR (5%), retention < 30 days (25%) and urethrolysis (12.5%). In comparison, 3 (7.5%) women undergoing low-stiffness SP sling had voiding dysfunction: high PVR (5%) and retention < 30 days (2.5%) (p<0.001). The incidence of voiding dysfunction was similar between high-stiffness and low-stiffness TO slings (NS). Fifteen percent of women undergoing high-stiffness TO sling had postoperative voiding dysfunction: high PVR (10%), retention <30 days (2.5%), and positional voiding (2.5%). In comparison, 12.5% of women had voiding dysfunction after low-stiffness TO sling: high PVR (5%), positional voiding (2.5%), retention < 30 days (2.5%), and urethrolysis (2.5%). Despite the differences in voiding dysfunction, QOL indices were significantly improved for all groups. CONCLUSIONS: We noted a significantly higher incidence of postoperative voiding dysfunction in women undergoing high-stiffness SP MUS, when compared with low-stiffness SP MUS. These slings may require even looser tensioning techniques than their low-stiffness counterparts. No significant difference was observed in highand lowstiffness TO slings, suggesting that this approach may be less dependent on specific tensioning technique.