AKT2, an oncogene encoding a protein serine-threonine kinase implicated in phosphatidylinositol-3-OH kinase signaling, is amplified in some human ovarian and pancreatic carcinomas. We previously demonstrated that the tumorigenicity and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cell lines with amplified AKT2 could be markedly reduced by transfection with antisense AKT2 constructs. To evaluate further the extent of AKT2 alterations in PDAC, DNA and immunohistochemical analyses were performed to assess amplification or overexpression of AKT2, respectively, in 72 PDACs. Thirty-five PDACs were subjected to Southern analyses, and AKT2 amplification was detected in seven tumors (20%). Forty-one formalin-fixed PDAC specimens were examined immunohistochemically with an anti-AKT2 monoclonal antibody, and moderate to intense staining was observed in eight tumors (20%). AKT2 immunostaining paralleled AKT2 genomic status in each of four cases in which both Southern and immunohistochemical analyses were performed. No obvious relationship was observed between AKT2 status and tumor TNM stage or grade. These observations suggest the utility of immunohistochemical analysis in assessing alterations of AKT2 in human cancers. Furthermore, the role played by the AKT2 kinase in the signaling pathways of various mitogenic growth factors implicated in the development of pancreatic cancer suggests that alteration of AKT2 may be an important component in the pathogenesis of a substantial subset of PDACs.

AKT2, an oncogene encoding a protein serine-threonine kinase implicated in phosphatidylinositol-3-OH kinase signaling, is amplified in some human ovarian and pancreatic carcinomas. We previously demonstrated that the tumorigenicity and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cell lines with amplified AKT2 could be markedly reduced by transfection with antisense AKT2 constructs. To evaluate further the extent of AKT2 alterations in PDAC, DNA and immunohistochemical analyses were performed to assess amplification or overexpression of AKT2, respectively, in 72 PDACs. Thirty-five PDACs were subjected to Southern analyses, and AKT2 amplification was detected in seven tumors (20%). Forty-one formalin-fixed PDAC specimens were examined immunohistochemically with an anti-AKT2 monoclonal antibody, and moderate to intense staining was observed in eight tumors (20%). AKT2 immunostaining paralleled AKT2 genomic status in each of four cases in which both Southern and immunohistochemical analyses were performed. No obvious relationship was observed between AKT2 status and tumor TNM stage or grade. These observations suggest the utility of immunohistochemical analysis in assessing alterations of AKT2 in human cancers. Furthermore, the role played by the AKT2 kinase in the signaling pathways of various mitogenic growth factors implicated in the development of pancreatic cancer suggests that alteration of AKT2 may be an important component in the pathogenesis of a substantial subset of PDACs.

The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and the urokinase receptor were examined in fetal and normal prostate tissues, benign prostatic hyperplasia and prostate cancer (n = 117). In situ hybridization with digoxigenin-labeled oligonucleotide probes demonstrated that TIMP-1 and TIMP-2 were expressed at elevated levels in the stroma of Gleason sum 5 tissues, whereas MMP-2 and MMP-9 were expressed at relatively low levels. In higher Gleason sum tissues (GS 8-10), TIMP-1 and TIMP-2 were not expressed, whereas MMP-2 and MMP-9 were intensely expressed. Furthermore, TIMP-1 and TIMP-2 expression was high in organ-confined specimens (OC, n = 43), somewhat lower in specimens with capsular penetration (CP, n = 29), and low or negative in samples with surgical margin/seminal vesicle (M/SV, n = 17) and lymph node (LN, n = 13) involvement. In contrast, MMP-2 and MMP-9 expression was low in the OC tissues; and noticeably higher in CP, M/SV, and LN specimens. Finally, correlation of TIMP and MMP expression with GS and pathological stage versus cure rate further revealed that a high percentage of organ-confined, GS 5 specimens expressing TIMP and little MMP were cured. In comparison, few of the GS 7-10 patients with capsular penetration and expressing MMP and little TIMP were cured. The data suggest that TIMP-1 (and TIMP-2) and MMP-2 (and MMP-9) are independent predictors of outcome.

BACKGROUND Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mutations in codon 12 of the K‐ras protooncogene is a frequent and early molecular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cigarette smoking and mutational activation of K‐ras in PDAC has been examined to a limited extent. The authors have examined the mutational status of K‐ras in nonneoplastic pancreata in relation to cigarette smoking status. METHODS Archival formalin fixed paraffin embedded specimens of nonneoplastic pancreata (n = 39) were obtained from the American Cancer Society and evaluated histopathologically. Specimens from age‐ and gender‐matched individuals were stratified into three groups: 1) those who never smoked cigarettes (n = 16), 2) those who smoked 1–2 packs/day for more than 20 years (n = 10 cases), and 3) those who smoked more than 2 packs/day for 20 or more years (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furthermore, none of the patients died of primary PDAC or had evidence of pancreatic metastases from an extrapancreatic primary tumor. Tissue sections were microdissected and deparaffinized, and genomic DNA was purified by standard proteinase K‐phenol‐chloroform extraction techniques. Genomic DNA was analyzed for mutations in codon 12 of the K‐ras protooncogene by two mutant‐allele‐enriched polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) assays and by multiplex PCR‐based ligase chain reaction (LCR) analyses. RESULTS Analyses of multiple microdissected pancreata specimens from 39 cases revealed wild‐type K‐ras codon 12 sequences in both nonsmoking individuals and those who smoked 1–2 packs/day for 20 or more years. K‐ras codon 12 mutations were confirmed by PCR‐RFLP and PCR‐LCR assays in 5 of 13 pancreata cases (39%) obtained from individuals who smoked more than 2 packs of cigarettes/day for 20 years or more (P < 0.005). The K‐ras mutation spectra revealed two G→T transversions, one G→C transversion and two G→A transitions. There was no clear relation between the incidence or spectra of mutations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atypia revealed reproducible K‐ras alterations. Similarly, among those 34 cases in which a wild‐type K‐ras sequence was revealed by both approaches, a similar histopathologic profile was evident. CONCLUSIONS Mutational activation of codon 12 of the K‐ras protooncogene was confirmed reproducibly by mutant allele‐enriched PCR‐RFLP and multiplex PCR‐LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from age‐ and gender‐matched individuals who smoked more than 2 packs of cigarettes/day for 20 or more years. The presence of a mutated or wild‐type or K‐ras was independent...

Background Engineered heart tissue (EHT) is being developed for clinical implantation in heart failure or congenital heart disease. The validity of the approach depends on the comprehensive functional characterization of EHT. Here we optimized the effects of modulating cell composition in self-organizing EHT and present detailed electrophysiological and contractile functional characterization. Methods EHT fibers with different cell densities (0.5×106 - 3×106) were generated from neonatal rat cardiac cells on a fibrin hydrogel scaffold. We measured contractile function using a force transducer and assessed force-length relationship, maximal force generation and rate of force generation (dF/dt). Using optical mapping with voltage sensitive dye, we assessed EHT fiber action potential (AP) and conduction velocity (CV) while transcript levels of myosin heavy chain beta (MyHβ) was measured by RT-PCR. Results We found a clear force-length relationship that was negatively impacted by increasing cell density. Maximal force generation and dF/dt were also significantly decreased with increasing cell densities. This decrease was not due to a selective expansion of non-contractile cells as MyHβ levels at endpoint reflected initial seeding density. EHT fibers with optimal cell density had an AP duration of 140.2 ms and a CV of 23.2 cm/s. Conclusion EHT display physiologically relevant features shared with native myocardium. Higher cell densities abrogate EHT contractile function, possibly due to sub-optimal cardiomyocyte performance in the absence of a functional vasculature. Finally, CV approaches that of native myocardium without any electrical or mechanical conditioning, suggesting that the self-organizing method may be superior to other rigid scaffold based EHT.