Bcl-xL is expressed in neuroblastoma cells and modulates chemotherapy-induced apoptosis.

bcl-x is a new member of the bcl-2 gene family and is highly expressed in neural tissues. The present study was designed to determine the expression of the bcl-x gene products in neuroblastoma (NB) and their role in the modulation of chemotherapy-induced apoptosis. Twenty-seven NB cell lines were screened by quantitative immunoprecipitation for Bcl-xL, Bcl-xS, and Bcl-2 expression. None of the cell lines expressed Bcl-xS. Twenty-four of 27 (88%) of the NB cell lines expressed Bcl-xL and 21 of 27 (78%) were positive for Bcl-2. The level of Bcl-xL and Bcl-2 expression was variable among the lines analyzed. Bcl-2 expression was restricted to cells of chromaffin lineage, whereas Bcl-xL was seen in both chromaffin and nonchromaffin lines. To determine whether Bcl-xL could mediate chemotherapy resistance, a NB cell line expressing negligible levels of Bcl-xL was transfected with a bcl-xL expression vector, and unique clones were generated expressing variable levels of Bcl-xL. Cells were treated either with cisplatinum (CP), 4-hydroperoxy-cyclophosphamide (4-HC), or etoposide (VP-16) to induce apoptosis, and cell viability and DNA degradation were determined. Following treatment with CP or 4-HC, Bcl-xL-expressing cells showed significantly increased viability as compared to vector-transfected controls (P < 0.005). Flow cytometric analysis of propidium iodide-stained nuclei following CP or 4-HC treatment revealed significantly increased DNA degradation in controls as compared to Bcl-xL-expressing lines (P < 0.004). DNA analysis by pulsed-field gel electrophoresis revealed high molecular weight (approximately 40 kb) DNA degradation in controls, whereas the DNA in cells expressing Bcl-xL was largely intact. In contrast to CP and 4-HC, results with VP-16 revealed a short-term delay in the onset of apoptosis in Bcl-xL-expressing cells with no long-term survival advantage. The results of these studies indicate Bcl-xL is expressed in NB cells and functions in a manner analogous to Bcl-2 by inhibiting chemotherapy-induced apoptosis.

Bcl-xS enhances adenoviral vector-induced apoptosis in neuroblastoma cells.

bcl-x is a member of the bcl-2 family of genes and by alternative splicing gives rise to two distinct mRNAs: bcl-xL and bcl-xS. We have previously investigated the expression of Bcl-x in neuroblastoma (NB) cell lines and have shown that Bcl-xL is expressed and functions to inhibit chemotherapy-induced apoptosis. However, none of the NB cell lines expressed Bcl-xS. The aim of the present study was to determine the effects of Bcl-xS expression on the viability of NB cells. A panel of NB cell lines (CHP-382, GOTO, SHEP-1, SHSY-5Y, and GI-CA-N) were infected with either a bcl-xS adenovirus (pAdRSV-bcl-xS) or a control virus (pAdRSV-lac-z). NB cells showed loss of viability with both viruses, although the bcl-xS virus was most toxic. Importantly, infection with the bcl-xS adenovirus resulted in rapid loss of cell viability, DNA fragmentation, and morphological features of apoptosis even in NB cells transfected to overexpress Bcl-2 and Bcl-xL. These findings suggest that deregulated expression of Bcl-xS using an adenovirus may provide a novel mechanism for initiating cell death in tumors that express Bcl-2 or Bcl-xL.

Bcl-2 and M-Myc coexpression increases IGF-IR and features of malignant growth in neuroblastoma cell lines.

The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB(2), Bcl-2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB) cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR).

Weaver syndrome with neuroblastoma and cardiovascular anomalies.

We report on an infant with Weaver syndrome, neoplasia and cardiovascular anomalies. Stage 4S neuroblastoma underwent spontaneous resolution. Three neoplasms have been reported in Weaver syndrome: another stage 4S neuroblastoma [Muhonen and Menezes, 1990: J Pediatr 116:596-599], an ovarian endodermal sinus tumor [Derry et al., 1999: J Med Genet 36:725-728], and a sacrococcygeal teratoma [Kelly et al., 2000: Am J Med Genet 95:492-495]. No case was associated with cardiovascular anomalies. Our patient had VSD and PDA, and although several other patients with Weaver syndrome have had cardiovascular anomalies, they were shown not to have neoplasia.

Fatal lymphoproliferative disease as a complication of Evans syndrome.

A 9-month-old boy had bruising and petechiae. Investigation revealed a Coombs-positive hemolytic anemia and immune-mediated thrombocytopenia. The infant was treated with intravenous immunoglobulin and steroids. The infant eventually had recurrent fevers, hepatosplenomegaly, pulmonary nodules, and parenchymal central nervous system (CNS) lesions develop. Results of a lung biopsy revealed a polyclonal lymphoproliferative disease. Polymerase chain reaction analysis showed the presence of the Epstein-Barr (EB) viral genome in the lung nodules. The infant died from progressive lung disease 6 months after the initial symptoms of Evans syndrome. Lymphoproliferative disease is known to occur in a variety of settings after immunosuppression, especially in solid organ transplant recipients. We report a case of polyclonal lymphocyte proliferation in a patient with Evans syndrome.

Congenital mesoblastic nephroma metastatic to the brain: a report of two cases.

Congenital mesoblastic nephroma was originally believed to be a universally benign neoplasm. More recently, aggressive congenital mesoblastic nephromas have been described with local recurrence and/or metastases. We report two patients with documented congenital mesoblastic nephroma which later metastasized to the brain.

Role of p53 in the regulation of irradiation-induced apoptosis in neuroblastoma cells.

Wild-type p53 plays a crucial role in the control of apoptosis following ionizing radiation (IR); conversely, mutant p53 is associated with IR resistance. Although wild-type p53 is expressed in virtually all neuroblastoma tumors, treatment failures secondary to inadequate local control with radiotherapy are a problem in patients with advanced stage disease. This apparent paradox is the focus of our interest. The Shep-1 neuroblastoma cell line is highly resistant to IR. This cell line contains a wild-type p53 gene and is an ideal model for studying the mechanism of IR resistance in this disease. Following high-dose IR, cell fractionation demonstrated that p53 is induced and targeted to the nucleus. The induced p53 is functional as p53-responsive genes (Waf-1 and MDM-2) are appropriately induced following IR. Intriguingly, overexpression of p53 could reverse the inherent IR resistance of Shep-1 cells. Multiple cell lines expressing variable levels of exogenous temperature-sensitive p53 were generated. Pulse induction of p53 alone did not affect Shep-1 cell viability, while induction of p53, followed by IR, resulted in cell death and DNA fragmentation proportional to the dose of IR and the level of p53 expression. These findings demonstrate that p53 overexpression renders Shep-1 cells IR-sensitive and suggest that large quantities of exogenous p53 can overcome the factors inhibiting p53-mediated, IR-induced apoptosis.

Is there a potential role for serum cardiac troponin I as a marker for myocardial dysfunction in pediatric patients receiving anthracycline-based therapy? A pilot study.

Serum cardiac troponin I (cTnI) levels have been reported to have high specificity and sensitivity to acute myocardial infarction and coronary ischemic syndromes in adult patients. Our goal was to evaluate the usefulness of serum cTnI in the early diagnosis of cardiac injury from anthracyclines, and to compare these values with echocardiographic findings of cardiac dysfunction. In this prospective study, children being treated on several Children's Cancer Group protocols underwent measurement of shortening fraction (SF), ejection fraction (EF), and serum cTnI levels prior to anthracycline therapy. Sequential serum cTnI levels were then measured along with regularly scheduled echocardiograms with progressively increasing doses of anthracyclines. Fifteen children with median age of 5.75 years (range, 15 months to 15.5 years) at diagnosis were evaluated. Anthracycline doses ranged from 11.72 mg/kg (in patients < 3 years of age) to 375 mg/m2. All but one patient had normal cTnI levels. His level measured at 1.7 ng/ml after 315 mg/m2, but was normal on follow-up testing. Initial SF ranged from 32 to 48%, and EF from 60 to 80%. On follow-up, SF and EF ranged from 30 to 41% and 55 to 70%, respectively. Both SF and EF were significantly lower (p < 0.001) as compared to the initial values. Despite this, all patients remained clinically asymptomatic from the cardiac standpoint. We did not observe elevations of serum cTnI levels in clinically asymptomatic children who received anthracycline therapy up to doses of 375 mg/m2. Does this mean that cardiac injury has not occurred? The possibility of assay sensitivity and the timing of serum sampling and echocardiograms may be important. In addition, larger sample size or longer follow-up may be helpful to determine if higher doses or symptomatic patients potentially have elevations in cTnI levels.

Keratinocytes derived from psoriatic plaques are resistant to apoptosis compared with normal skin.

Previously we observed that hyperplastic epidermal keratinocytes characteristic of psoriasis had abundant amounts of the cell survival protein Bcl-xL; however, whether this overexpression correlated with enhanced survival was unclear because the majority of epidermal cells possess nuclei that are positively labeled by an assay typically regarded as indicative of cells undergoing apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining). To clarify this apparent discrepancy, we explored the propensity of keratinocytes derived from psoriatic plaques to undergo apoptosis and also determined the reliability of TUNEL staining as an indicator of apoptosis in keratinocytes in vitro and in vivo. First, a keratinocyte cell line, HaCat, was examined before and after being suspended in semisolid medium (methylcellulose) using flow cytometry to detect TUNEL-positive cells, and the percentage of positive cells was correlated to the presence or absence of double-stranded DNA fragmentation using pulsed field gel electrophoresis. After 18 hours in methylcellulose suspension, apoptosis was detected in HaCat cells when at least 5% of the cell population was undergoing programmed cell death. Second, we examined 23 clinical specimens of skin (13 from psoriatic patients and 10 from healthy control subjects) and observed that no double-stranded DNA fragmentation was present in any of the freshly isolated keratinocytes from either normal or psoriatic patients. Keratinocytes from 9 of 12 normal skin samples underwent double-stranded DNA fragmentation after being in methylcellulose for 18 to 24 hours, which contrasts with keratinocytes from lesions of psoriasis where only 1 of 13 of the skin samples had these changes. Third, two-color immunofluorescence staining of psoriatic plaques revealed that numerous TUNEL-positive keratinocytes were also positive for proliferating cell nuclear antigen and Ki-67 antigens and that by flow cytometry TUNEL-positive keratinocytes obtained from psoriatic plaques possessed a DNA content profile indicative of proliferating and not dying cells. These results demonstrate that keratinocytes within psoriatic plaques do not have double-stranded DNA breaks, that they have a prolonged capacity to resist induction of apoptosis compared with normal-skin-derived keratinocytes or cultured HaCat cells, and that caution is necessary for proper interpretation related to detection of 3'-OH DNA ends (ie, TUNEL positivity) in skin, as it can be associated with DNA synthesis as well as cell death.