p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic carcinoma cell line LNCaP.

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.

Fas-mediated apoptosis in human prostatic carcinoma cell lines occurs via activation of caspase-8 and caspase-7.

We previously demonstrated that treatment with cycloheximide (CHX) converted the phenotype of Fas-resistant human prostatic carcinoma cell lines to Fas-sensitive and that resistance to Fas-mediated apoptosis was due to a dominant-negative protein(s). In this study, we investigated the sequential activation of caspase family members, to gain insight into the likely site of action of the suppressor protein(s). We did not find Tyr-Val-Ala-Aspase activity in any of the cell lines examined. Time-dependent Asp-Glu-Val-Aspase activity was detected during Fas-mediated apoptosis in Fas-sensitive cell lines PC3 and ALVA31. Asp-Glu-Val-Aspase activity in Fas-resistant cell lines DU145 and JCA1, was detected only under combined treatment with CHX and anti-Fas agonistic mAb. In experiments with caspase inhibitors we show that Fas-mediated apoptosis in PC3 is mainly executed by the caspase-3 subfamily, but another member(s) of the caspase family may be involved in Fas-mediated apoptosis in ALVA31, DU145, and JCA1. Western blot analysis revealed that Fas-ligation activated caspase-7, but not caspase-3. The activated form of caspase-8 was detected in DU145 only after 4 h of simultaneous treatment with CHX and anti-Fas mAb, whereas in PC3 caspase-8 was found to be activated after 1 h of Fas-ligation. We have also found that treatment with staurosporin did not activate caspase-8, whereas staurosporin induced apoptosis at the same levels in both Fas-resistant and Fas-sensitive cell lines. These results suggest that an inhibitory protein(s), which suppresses apoptosis in Fas-resistant cell lines, presumably acts at the apex of apoptotic cascade by preventing the activation of caspase-8.

Dominant nature of the resistance to Fas- and tumor necrosis factor-alpha-mediated apoptosis in human prostatic carcinoma cell lines.

We have recently found (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997) that, although Fas ligation induced apoptosis in two of six human prostatic carcinoma cell lines investigated, the apoptotic machinery involved in Fas-mediated killing is already in place in Fas-resistant cell lines. Here, we investigated Fas- and tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis in cell hybrids between resistant (DU145 and JCA1) and sensitive (ALVA31 and PC3) cell lines. All three types of hybrid cells investigated, F1(DU145 x PC3), F1(JCA1 x PC3), and F1(JCA1 x ALVA31), were found to be resistant to Fas- and TNF-alpha-mediated apoptosis at the same level as the corresponding parental resistant cell lines. These results indicate that resistance to Fas- and TNF-alpha-mediated apoptosis dominates over sensitivity in cell hybrids and suggest that resistance may be regulated by an apoptosis suppressor factor or factors acting in resistant but not in sensitive cells.

Fas-mediated apoptosis in human prostatic carcinoma cell lines.

Of six prostatic carcinoma cell lines examined (ALVA31, DU145, JCA1, LNCaP, ND1, and PC3) by flow cytometric analysis, all were found to be positive for Fas antigen. Furthermore, of the prostate tissue specimens studied (six cases), all revealed Fas expression in benign and malignant epithelial cells. The agonistic anti-Fas monoclonal antibody (IPO-4) induced apoptosis in only two of six cell lines investigated, PC3 and ALVA31. PCR analysis indicated that all cell lines expressed normal transmembrane and death domains of Fas antigen. Using Western blot analysis, we found abundant expression of p53 in the cytoplasm of two Fas-resistant cell lines, DU145 and ND1, and did not find p53 in two Fas-sensitive cell lines, PC3 and ALVA31. Western blot and PCR analysis did not show consistent differences between cell lines examined in the expression of Bcl-2, Bcl-X(L), Bcl-X(S), and Bak. In contrast, Bax protein was not detected in two Fas-resistant cell lines, DU145 and ND1. We also showed that three Fas-resistant cell lines, DU145, ND1, and JCA1, expressed CD40, whereas the two Fas-sensitive cell lines, PC3 and ALVA31, were CD40 negative. Fas-sensitive cell lines were transfected with the cDNA encoding CD40, and the CD40-positive transfectant became more resistant to growth inhibition mediated by treatment with TNF-alpha and anti-Fas monoclonal antibody. Treatment with cycloheximide converted the phenotype of resistant cell lines from Fas resistant to Fas sensitive. Moreover, anti-Fas treatment of both resistant and sensitive cell lines induced rapid tyrosine phosphorylation or dephosphorylation of multiple proteins. These results suggest that the apoptotic machinery involved in DNA fragmentation is already in place in Fas-resistant cell lines, and thus, Fas-mediated apoptosis could be a target for therapeutic intervention.

Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group.

OBJECTIVE: To determine whether a nutritional supplement of selenium will decrease the incidence of cancer. DESIGN: A multicenter, double-blind, randomized, placebo-controlled cancer prevention trial. SETTING: Seven dermatology clinics in the eastern United States. PATIENTS: A total of 1312 patients (mean age, 63 years; range, 18-80 years) with a history of basal cell or squamous cell carcinomas of the skin were randomized from 1983 through 1991. Patients were treated for a mean (SD) of 4.5 (2.8) years and had a total follow-up of 6.4 (2.0) years. INTERVENTIONS: Oral administration of 200 microg of selenium per day or placebo. MAIN OUTCOME MEASURES: The primary end points for the trial were the incidences of basal and squamous cell carcinomas of the skin. The secondary end points, established in 1990, were all-cause mortality and total cancer mortality, total cancer incidence, and the incidences of lung, prostate, and colorectal cancers. RESULTS: After a total follow-up of 8271 person-years, selenium treatment did not significantly affect the incidence of basal cell or squamous cell skin cancer. There were 377 new cases of basal cell skin cancer among patients in the selenium group and 350 cases among the control group (relative risk [RR], 1.10; 95% confidence interval [CI], 0.95-1.28), and 218 new squamous cell skin cancers in the selenium group and 190 cases among the controls (RR, 1.14; 95% CI, 0.93-1.39). Analysis of secondary end points revealed that, compared with controls, patients treated with selenium had a nonsignificant reduction in all-cause mortality (108 deaths in the selenium group and 129 deaths in the control group [RR; 0.83; 95% CI, 0.63-1.08]) and significant reductions in total cancer mortality (29 deaths in the selenium treatment group and 57 deaths in controls [RR, 0.50; 95% CI, 0.31-0.80]), total cancer incidence (77 cancers in the selenium group and 119 in controls [RR, 0.63; 95% CI, 0.47-0.85]), and incidences of lung, colorectal, and prostate cancers. Primarily because of the apparent reductions in total cancer mortality and total cancer incidence in the selenium group, the blinded phase of the trial was stopped early. No cases of selenium toxicity occurred. CONCLUSIONS: Selenium treatment did not protect against development of basal or squamous cell carcinomas of the skin. However, results from secondary end-point analyses support the hypothesis that supplemental selenium may reduce the incidence of, and mortality from, carcinomas of several sites. These effects of selenium require confirmation in an independent trial of appropriate design before new public health recommendations regarding selenium supplementation can be made

Nociceptive role of substance-P in the knee joint of a patient with congenital insensitivity to pain.

This case report describes the immunocytochemical examination of tissue from a 9-year-old black child diagnosed with congenital insensitivity to pain at age 5. A recent fall and resulting patella fracture required surgical treatment. Biopsies of the distal pole and surrounding soft tissue, as well as a sample of his patellofemoral joint fluid, were taken at the time of partial patellectomy and analyzed for substance-P (SP). Morphologic staining of the patella showed a grossly degenerated patellofemoral articular surface. Examination of tissue sections stained either immunocytochemically with diaminobenzidine DAB or by a rhodamine fluorescent labeling technique showed no evidence of SP-positive nerve fibers. Furthermore, only a trace amount of SP (7.29 pg/ml) was detected in a sample of the patient's knee joint synovial fluid. This patient's absence of pain sensation in conjunction with the absence of SP nerve fibers in stained patella sections and identification of only trace levels of SP in his synovial fluid, further implicates this neuropeptide in nociceptive innervation of diarthrodial joints.

Substance P innervation of lumbar spine facet joints.

Sixteen adult human lumbar spine facet joints were harvested from patients undergoing various lumbar spine procedures. Diagnoses included degenerative disc disease, adult spinal deformity, facet joint degenerative arthritis, and degenerative spondylolisthesis. Facet joints were processed for routine hematoxylin and eosin staining. Immunohistochemical analysis was performed using a monoclonal antibody to substance P. All facets grossly exhibited evidence of degenerative disease, including cartilage surface irregularity and fibrillation. Histological examination of facets obtained from patients with degenerative spinal conditions demonstrated erosion channels extending through the subchondral bone and calcified cartilage into the articular cartilage. Immunostaining showed the presence of substance P-positive nerve fibers within these erosion channels, and also within marrow spaces. The presence of substance P nerve fibers within subchondral bone of degenerative lumbar facet joints implicates this type of joint in the etiology of low back pain.

An unusual presentation of secondary syphilis in a patient with human immunodeficiency virus infection. A case report and review of the literature.

BACKGROUND: Syphilis has been reported to assume unusual clinical appearances and to exhibit unusual courses in patients infected with the human immunodeficiency virus (HIV) type 1. We recently observed a distinct manifestation of syphilis in an HIV-infected patient with features not previously described. OBSERVATIONS: A 38-year-old HIV-seropositive homosexual man presented with fever, chills, malaise, and a cutaneous eruption consisting of indurated, shiny, erythematous plaques that were confluent on the face and scalp leading to alopecia and extreme tautness of the skin. Initial clinical diagnoses included lymphoreticular malignancy and infection. Although cultures yielded Staphylococcus aureus, a skin biopsy specimen was diagnostic of syphilis. CONCLUSIONS: This case demonstrates an unusual clinical manifestation of syphilis in a patient with HIV infection and emphasizes the importance of considering cutaneous secondary syphilis in the differential diagnosis of virtually any inflammatory cutaneous disorder in HIV-seropositive individuals.

Histamine release from rodent and human mast cells induced by protoporphyrin and ultraviolet light: studies of the mechanism of mast-cell activation in erythropoietic protoporphyria.

We report that protoporphyrin (PP) and ultraviolet light (UVA) induces histamine release from rat peritoneal mast cells, mouse bone marrow mast cells and human cutaneous mast cells in a dose- and temperature-dependent manner. The mast-cell activation was associated with loss of membrane integrity and inhibited by the hydrogen peroxide scavenger, catalase. Histamine release was independent of extracellular calcium in the rodent mast cells, but was markedly reduced in the absence of calcium in human cells. These findings indicate that PP and UVA induce mast-cell-mediator release by a process that may involve hydrogen peroxide formation. There appear to be differences in response to PP and UVA between rodent and human mast cells.

Innervation of the human knee joint by substance-P fibers.

Anterior knee pain is a frequent musculoskeletal complaint affecting all ages, both sexes, athletes, and nonathletes alike. Numerous theories have been proposed regarding its etiology including patellar malalignment, quadriceps insufficiency, subluxation, quadriceps muscle tightness, and chondral defects. However, the mechanism by which these factors produce this pain is not clear. Knowledge of the distribution of nociceptive nerve fibers around the knee would seem to provide insight in treating these painful conditions. Eleven human patellae--eight specimens from patients with degenerative patellofemoral disease and three normals--were evaluated. Immunohistochemical techniques using monoclonal antibody to substance-P were employed to identify nociceptive fibers. Substance-P is a nociceptive neurotransmitter found in afferent nerve fibers. Substance-P fibers were isolated in the retinaculum, fat pad, periosteum, and subchondral plate of patellae affected with degenerative disease. This study demonstrates that selective tracting of nociceptive pain fibers is possible around the knee both in soft tissue and, in some circumstances, bone. The subchondral plate of normal patellae did not demonstrate erosion channels, but those with chondral defects from degenerative disease did. Nociceptive fibers found in these defects may explain the origin of symptoms in some patients. The distribution of substance-P nerve fibers in the soft tissues around the knee suggests that denervation may be the mechanism by which surgical procedures for anterior knee pain produce favorable results.

Genetic prenatal aqueductal stenosis with hydrocephalus in rat.

A recessive mutation which arose in Wistar albino rats was variably expressed in the homozygous state as prenatal stenosis of the aqueduct with resultant hydrocephalus. The condition was often compatible with survival to adulthood and with successful reproduction. Mildly sparse hair was the constant gene marker. Eye defects and sometimes foot deformities occurred. The first observable ultrastructural alteration was a disruption of the integrity of the neuroepithelial basal lamina in the cephalic neural tube of affected embryos as early as the 11th fetal day (16-24 somite pairs). The hydrocephalic syndrome closely resembled that produced by giving folic acid analogs to, or producing vitamin B12 deficiency in, pregnant rats in the period including the 11th day. Neither vitamin B12 nor folate, nor certain metabolites closely related to their metabolism, prevented the gene's expression. Homozygote mutants mated with homozygote mutants produced 70% hydrocephalic (dome-shaped heads) offspring, but if the mother was heterozygote, there was a "protective" effect and the number of hydrocephalic young was disproportionately smaller.

A new cell surface relationship between neuroepithelial cells during rat neural tube development.

Electron microscopic examination of the developing neural tube in 11th to 13th day rat fetuses revealed a new cell surface relationship between differentiating neuroepithelial cells. Cytoplasmic projections possessing terminal dilations were observed extending from neuroepithelial cells through cytoplasmic furrows into large coated pits at the surface of adjacent cells. This cell-to-cell relationship provides a mechanism for the internalization of surface molecules and possibly even cytoplasmic constituents. Communication between donor and recipient cells mediated in this way suggests a route for the sharing of macromolecules, including cytoplasmic fragments, which could function as regulators in embryonic development and differentiation.

Alpha-aminoisobutyric acid uptake in primary cultures of astrocytes.

Homotypically pure cultures of rat brain astrocytes were used to examine some aspects of non-neuronal A-system (alanine preferring) amino acid uptake. The A-system specific probe, alpha-aminoisobutyric acid is transported rapidly, and a steady state distribution ratio of 9-25 is reached after 30 minute incubations. Kinetic estimates derived from uptake progress curves indicated a Km of 1.35 mM and a Vmax of 133 nmol/min/mg protein. Uptake is reduced in the absence of either Na+ or K+. Elevations in extracellular K+, a putative metabolic modulator of neuroglia, did not affect uptake.

Beta-alanine uptake is not a marker for brain astroglia in culture.

The properties of the beta amino acid transport system were examined in cultivated rat brain astrocytes, using the specific probe, beta-alanine. The uptake of beta-alanine is thought to be glial specific. Beta-alanine was not actively transported and the intracellular accumulation was not altered by coincubation with GABA, alanine, glutamate, or methionine. We suggested therefore that beta-alanine is passively taken up by a mechanism distinct from the transport system for GABA.

Potassium modulation of methionine uptake in astrocytes in vitro.

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 microM. At steady state conditions, methionine is concentrated 30-50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 microM. We recently reported that methionine uptake is decreased by elevations in extracellular K+. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5 microM, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K+ equal to 6.9 mM, the apparent Vmax of methionine transport is 182 pmol/min/mg protein, and the Km is 1.3 microM. Where K+ is shifted to 11.9 mM, the Km remains unchanged, and the Vmax is reduced by half.

Chronological changes in acid phosphatase activity within neurons and perineuronal satellite cells of the inferior vagal ganglion of the cat induced by vagotomy.

The hexazonium pararosaniline method was employed to describe the distribution of acid phosphatase activity, chronologically, within neurons and their investing satellite cells of the inferior vagal ganglion of the cat after vagotomy. In control ganglia, acid phosphatase activity was invariably confined to the cytoplasm of neurons and satellite cells. Reaction product was visible as distinct granules within neuronal perikarya. The cytoplasm of perineuronal satellite cells also contained reaction product but, in most instances, activity was weak and granules were difficult to distinguish. No reaction product was observed in myelin or axonal processes; nuclear staining was absent. Acid phosphatase activity was increased in ganglionic neurons as early as 24 hours after vagotomy. Increased activity in perineuronal satellite cells was not evident until 3 days post-operatively. By 15 days, activity was ubiquitously increased in the cytoplasm of both neurons and satellite cells. Evidence suggesting neuronophagia was also apparent. Between 30 and 60 days post-operatively acid phosphatase activity gradually decreased in both neurons and satellite cells until a picture comparable with that seen in control tissue sections was visible. The functional significance of these changes in acid phosphatase activity within an altered metabolic environment induced by vagotomy is discussed.

Propagation and histological characterization of a homotypic population of astrocytes derived from neonatal rat brain.

Tissue derived from the cerebra of 3-4 day old rats was minced, trypsinized and placed in tissue culture medium. Cells attained a monolayer in twelve to fourteen days when plated at a density of 10(2) cells/cm2. Selected culturing procedures designed to inhibit neuronal growth while encouraging glial growth yielded monolayers which consisted almost exclusively of astrocytes. Cells were identified using established morphological criteria for brain cells in culture. These observations were supplemented using certain histological and histochemical techniques frequently employed in investigative work for the identification of glial cells, particularly astrocytes. Results of all the above procedures indicate cell cultures which apparently consist exclusively of astrocytes. The preparation of such a homotypic population of normal, viable astroglial cells, derived from an animal universally used in investigative work on the nervous system, is perceived as an important contribution to the future study of glial characteristics and their interactions at morphological and functional levels.