Kenneth D. Somers

Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition

Pulse power technology using high intensity (up to 300 kV/cm) nanosecond pulsed electric fields (nsPEF) has been applied for decontamination and amelioration of biofouling, but until now effects on human cells have not been investigated. To analyze structural and functional changes in human cells and solid tumors following exposure to nsPEF we utilized flow cytometry and immunofluorescence microscopy. We provide further support for the hypothesis that as the pulse duration is decreased, there is a lower incidence of electric field interactions at the plasma membrane and a higher incidence of interactions with intracellular structures. The nsPEF effects are pulse duration/electric field intensity-dependent and energy density- or temperature-independent. We also show that nsPEF induces programmed cell death (apoptosis) in cultured cells as indicated by cell shrinkage, Annexin-V-FITC binding to phosphatidylserine on intact cells, and caspase activation. Mouse fibrosarcoma tumors exposed to nsPEF exhibit fragmented DNA and reduced tumor growth in a mouse model. These studies show that nsPEF effects are distinctly different than electroporation pulses and provide the first evidence for the potential application of nsPEF to induce apoptosis and inhibit tumor growth.

Diverse Effects of Nanosecond Pulsed Electric Fields on Cells and Tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

Fibrin Deposition in Peyronie's Disease Plaque

PURPOSE Peyronies disease is a pathological fibrosis characterized by excessive deposition of collagen in the plaque. Although the etiology of Peyronies disease is unknown, trauma has been hypothesized as the inciting event. In an effort to obtain more insight into the pathogenesis of Peyronies disease plaque tissue was examined for collagen, elastic fiber, and fibrin content and distribution. MATERIALS AND METHODS Plaque tissue specimens from 33 patients with Peyronies disease, control penile tissue and nodular tissue from 8 patients with Dupuytrens contracture were analyzed histochemically for collagen staining and elastic fiber structure and distribution. Plaque tissue from 19 Peyronies disease patients, control tissue and nodular tissue from Dupuytrens disease were also analyzed for the presence of fibrin by histochemical staining and immunoblotting. RESULTS Aberrantly stained collagen was detected in 32 of 33 plaque specimens (97%) and disrupted elastic fibers in 31 of the same specimens (94%). Fibrin deposition was detected histochemically in plaque tissue from 18 of 19 patients (95%) but it was not detectable in normal or scarred tunica from control patients. The presence of authentic fibrin accumulation in plaque tissue was confirmed by immunoblot analysis but fibrin was not detected in dermal tissue extracts from the same patient. Aberrant collagen staining and fibrin deposition were detected in nodular tissue from 7 of 8 Dupuytrens contracture patients (88%) and altered elastic fibers in 5 of the same patients (63%). CONCLUSIONS Deposition of fibrin in plaque tissue is consistent with the hypothesis that repetitive microvascular injury results in fibrin deposition in the tissue space and has served to provide insights into the pathophysiology of Peyronies disease. We propose a model that accounts for the clinical and biological features of Peyronies disease.

Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro.

Cortactin, a p80/85 protein first identified as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, we stably overexpressed cortactin in NIH3T3 fibroblasts and evaluated the effects of elevated cortactin on cellular proliferation, motility and invasiveness. Cortactin overexpressing cells did not display any striking morphological changes, nor any significant differences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells were anchorage dependent for growth. Interestingly, cortactin overexpressing cells were more motile and invasive in modified Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by influencing tumor cell migration and invasion.

Serum Protein Profiles to Identify Head and Neck Cancer

Purpose: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. Experimental Design: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry ProteinChip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). Results: Several proteins, with masses ranging from 2,778 to 20,800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. Conclusions: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.

Isolation and Characterization of Collagen in Peyronie’s Disease

Peyronies disease is characterized histologically by excessive collagen deposition in the lesion. We examined the collagen types in Peyronies disease plaque tissues compared to unaffected tissues from the same patient, other control tissues, and Dupuytrens contracture. Gel electrophoresis of pepsin-solubilized collagen demonstrated the presence of type I collagen and an increased content of type III collagen in plaque tissue. Increased type III collagen was detected in apparently normal tissue adjacent to the plaque and in Dupuytrens lesion, confirming previous findings. Although the cause of excessive collagen accumulation of Peyronies disease is unknown, the results suggest an imbalance in the regulation of extracellular matrix production leading to pathologic fibrosis.

Calcium signaling in prostate cancer cells : Evidence for multiple receptors and enhanced sensitivity to bombesin/GRP

Cellular calcium is an important second messenger for growth regulation. We sought to identify potentially important receptors on prostate tumor cells by screening over 20 agonists for their ability to increase intracellular free calcium ([Ca2+]i) in several human prostate tumor cell lines.

Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines

Aberrant cellular signaling is a central feature of malignant cells and a potential target for anti‐cancer therapy. Carboxy‐amido‐triazole (CAI) is a calcium influx inhibitor that alters calcium‐sensitive signal transduction pathways and suppresses the proliferative and metastatic potential of malignant cells. We have examined the effects of CAI on several tumor‐associated parameters in human prostate cancer cell lines to evaluate the potential of CAI as a signal‐transduction therapy agent for advanced‐stage prostate cancer. Measuring anchorage‐dependent cell growth, continuous application of CAI inhibited the growth of DU‐145, PPC‐1, PC3 and LNCaP tumor cells with 50% inhibitory concentrations ranging 10–30 μM. Direct cell enumeration assays revealed that the growth‐suppressing activity of CAI toward DU‐145 cells was reversible, indicating a cytostatic effect of the drug on tumor cells. The drug also inhibited the proliferation of several immortalized human prostatic epithelial cell lines. The proliferation of HaCaT‐ and RHEK‐1‐immortalized keratinocyte cell lines was relatively insensitive to CAI. Additionally, invasion by DU‐145, PC3 and PPC‐1 cells through Matrigel in vitro was reduced approximately 60–70% by 10 μM CAI. Other cellular effects of CAI included an attenuation of the elevation of intracellular free calcium in response to bombesin and carbachol in PC3 cells and a marked dose‐dependent inhibition of prostate‐specific antigen secretion in LNCaP cell cultures.

Chromosome abnormalities in Peyronie's disease.

Peyronies disease is a localized and progressive fibrosis of unknown etiology that affects the tunica albuginea of the penis. We examined cytogenetically cell cultures derived from plaque, adjacent tunica, dermis and lymphocytes in patients with Peyronies disease, and compared the results to cell cultures established from the tunica albuginea of control patients. Chromosomal abnormalities were detected in 9 plaque-derived cell cultures from 7 of 12 Peyronies disease patients (58 per cent). Cells cultured from adjacent tunica, dermis and lymphocytes from the same patients were karyotypically normal, as were cultures derived from control (chordee and penile scar) patients. Chromosomal aberrations consisted of 5 numerical changes and 4 structural rearrangements, and included chromosomal additions (trisomy 7 and trisomy 8), deletions (45X,-Y), reciprocal translocations and inversions or markers. In 2 instances cultures derived from plaque tissue contained 2 independent chromosomal abnormalities. The apparently random chromosomal changes associated with Peyronies disease suggests that karyotypic instability may be a common feature of cells within the plaque. It presently is unclear whether this finding represents multiple pathways for the development of Peyronies disease or secondary consequences of Peyronies disease.

Cytogenetic analysis of four primary prostatic cultures

Primary cell cultures were established from tissue specimens obtained from patients undergoing transurethral resection of the prostate. Cytogenetic analysis of these cultures revealed a normal male chromosomal complement from one and a 45,X karyotype from another patient with benign prostatic hyperplasia. In addition, a normal male chromosomal complement was observed from a moderately differentiated prostatic carcinoma, and a grossly abnormal karyotype was observed from a poorly differentiated adenocarcinoma of the prostate. This latter specimen contained a modal chromosome number of 84 with several consistent marker chromosomes including homogeneous staining regions and double minutes, and no normal chromosomes 3, 5, 10, 15 or Y. Primary prostatic cell cultures exhibit epithelial-specific keratin intermediate filament proteins, and, in conjunction with cytogenetic analysis, provide a model for the study of human prostate cancer.