Functional analysis of the host defense peptide Human Beta Defensin-1: new insight into its potential role in cancer.

Although it is known that innate immunity is key for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have anti-tumor activity. Human Beta Defensin-1 (hBD-1), an important component of the innate immune response, is lost at high frequencies in malignant prostatic tissue, while high levels of expression are maintained in adjacent benign regions. In prostate carcinoma, frequent genetic alterations occur in the 8p22-23 region and several studies indicate there may be multiple tumor suppressor genes present within this region. The high incidence of loss of hBD-1 expression in prostate cancer, along with its chromosomal location of 8p23.2, raised the possibility that it may play a role in tumor suppression. To gain insight as to its function in prostate cancer, hBD-1 was cloned and ectopically expressed in four prostate cancer cell lines. Induction of hBD-1 expression resulted in a decrease in cellular growth in DU145 and PC3 cells. However, hBD-1 has no effect on the growth of androgen receptor (AR) positive LNCaP prostate cancer cells, but was again growth suppressive to PC3 cells with ectopic AR expression (PC3/AR+). hBD-1 also caused rapid induction of cytolysis and caspase-mediated apoptosis in DU145 and PC3 prostate cancer cells. Although the regulation of hBD-1 was not addressed in this study, our preliminary data demonstrated that the pathways involved may include cMYC and PAX2. Data presented here are the first to provide evidence of its potential role in prostate cancer cell death.

Identification and characterization of an L-type Cav1.2 channel in spiral ligament fibrocytes of gerbil inner ear.

Intracellular free Ca2+ levels are critical to the activity of BK channels in inner ear type I spiral ligament fibrocytes. However, the mechanisms for regulating intracellular Ca2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca2+ channel in type I spiral ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were -40 and -6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4 +/- 76.2 pA (n = 19); 10(-6) M nifedipine significantly inhibited the inward currents by 90.3 +/- 1.2% (n = 11). RT-PCR analysis revealed that cultured type I spiral ligament fibrocytes express the alpha1C isoform of the L-type Ca2+ channels encoded by the Cav1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated spiral ligament tissues. The Cav1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca2+ levels in type I spiral ligament fibrocytes, and thus modulate BK channel activity in these cells.

BK channels mediate the voltage-dependent outward current in type I spiral ligament fibrocytes.

Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K(+) recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I spiral ligament fibrocytes (SLFs), a major component of the K(+) recycling pathway, have a dominant K(+) membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70+/-1.2 mV at 1 nM intracellular free Ca(2+) and shifted to 38+/-0.2 mV at 20 microM intracellular free Ca(2+) (n=4-6). The reversal potential of whole-cell tail currents against different bath K(+) concentrations was 52 mV per decade (n=3-6). The sequence of relative ion permeability of the whole-cell conductance was K(+)>Rb(+)z.Gt;Cs(+)>Na(+) (n=5-17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC(50) values of 0.07 mM and 0.013 microM, respectively (n=3-7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K(+)-selective and sensitive to IbTx (n=4-9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be approximately 6 s with repetitive voltage pulses from -70 to 80 mV (n=3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K(+) movement from perilymph to the stria vascularis.

Functional and gene expression analysis of the p53 signaling pathway in clear cell sarcoma of the kidney and congenital mesoblastic nephroma.

Mutation of p53 has been implicated in progression of classical Wilms tumor (WT) into the anaplastic variant (AWT), drug resistance, and poor prognosis. Because of prognostic similarities, clear cell sarcoma of the kidney (CCSK) has been classified with AWT and other aggressive pediatric renal tumors, apart from congenital mesoblastic nephroma (CMN), which is instead a relatively benign tumor of neonates. Initially, CCSK and CMN were assumed to be ontologically related, but the role of p53 in the pathogenesis of either disease has not been sufficiently evaluated as in AWT. We examined the status of p53 in CMN and CCSK in comparison to AWT by immunohistochemistry and mRNA analysis of p53, the downstream effector p21(WAF-1/CIP-1) ( p21), the multidrug resistance gene MDR-1, a putative target of p53, and the p53-antagonist Mdm-2. Surprisingly, strong p53 nuclear immunoreactivity was found in cultures from two CMN specimens, but not in frozen or fixed tumor tissue from five other CMN specimens, nor in cell lines or tumor tissue from CCSK. Sequence analysis excluded p53 mutations. The size of the p53 mRNA in CMN and CCSK primary tumors excluded gross deletions or rearrangements. Low levels of Mdm-2 mRNA in CCSK and CMN primary tumors and cultures did not support a role for Mdm-2. Absence of MDR-1 mRNA excluded MDR-1 in the drug-resistant phenotype of CCSK. Cisplatin-induced p21 transactivation assays and G(1) cell cycle arrest analyses showed that p21 transactivation and G(1) arrest occurred in both CCSK and CMN cultures, demonstrating integrity of the p53 signal transduction pathway. Absence of p53 functional abnormalities excluded relationships between CCSK and CMN as in AWT, supporting the association of cellular CMN with congenital fibrosarcomas as more recently proposed.

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats.

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram-centimeter force (g-cm) injury was induced in rats by a weight-drop method and allowed to progress for 4 hr. One-centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2-AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca(2+)) levels following injury, compared with corresponding tissue sections from sham-operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double-immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl-2 ratio, indicating the induction of the mitochondria-mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double-immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca(2+) levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.

Nephropathy in a hypercholesterolemic mouse model with streptozotocin-induced diabetes.

BACKGROUND/AIMS: The contribution of preexisting hypercholesterolemia to diabetic nephropathy remains unclear. We assessed the impact of hypercholesterolemia on diabetic nephropathy using a double knockout (DKO) mouse, null for the low-density lipoprotein receptor (LDLRNDASH;/NDASH;) and the apoB mRNA editing catalytic polypeptide 1 (APOBEC1NDASH;/NDASH;). METHODS: Wild-type (WT) and DKO mice received sham or streptozotocin injections at age 7 weeks, yielding control (WT-C, DKO-C) and diabetic (WT-D, DKO-D) groups. At sacrifice (age 40 weeks), albuminuria was determined by ELISA, and kidney sections were examined by light and electron microscopy. RESULTS: Albuminuria increased in diabetic mice (WT-D: 82.4 +/- 37.2 microg/18 h; DKO-D: 58.0 +/- 45.7 microg/18 h) versusnondiabetic controls (WT-C: 10.2 +/- 7.2 microg/18 h; DKO-C: 8.6 +/- 5.3 microg/18 h) (p LT; 0.0001), but was unaffected by hypercholesterolemia. Light microscopy of kidney sections demonstrated increased collagen levels in glomeruli in WT-D mice, but not in DKO-D mice or either control group. Electron microscopy showed a thickened glomerular basement membrane in WT-D mice only. The proximal tubular basement membrane thickness was increased in both diabetic groups versusnondiabetic controls (p LT; 0.01); in WT-D mice this was attributable to collagen accumulation, but in DKO-D mice it was mainly caused by lipid vacuoles. CONCLUSIONS: In this animal model, preexisting hypercholesterolemia did not exacerbate either glomerular lesions of diabetes (collagen accumulation, basement membrane thickening) or albuminuria, but appeared to mitigate these effects. Furthermore, the combination of hypercholesterolemia and diabetes resulted in a significant lipid accumulation in the tubular basement membrane.

Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase.

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.