Emil Bogenmann

Expression of trkA cDNA in neuroblastomas mediates differentiation in vitro and in vivo

The human trkA cDNA was transfected into a malignant human neuroblastoma (NB) cell line (HTLA230) to investigate its role in NB growth and differentiation. This cell line lacks expression of both endogenous trkA and gp75NGFR genes. Transfectants expressing the trkA mRNA and surface-bound receptors transcriptionally activate immediate-early genes (c-fos, c-jun, and jun-B) following nerve growth factor (NGF) stimulation. NGF treatment induces growth arrest as well as down-regulation of the amplified N-myc oncogene. Genes selectively expressed in mature neurons (SCG-10, ret proto-oncogene, GAP-43, etc.) are transcriptionally activated, and neurite outgrowth further demonstrates differentiation of transfectants following NGF stimulation. trkA-expressing NB cells remain tumorigenic in nude mice; however, subcutaneous treatment of tumor-bearing mice with NGF induces Schwannian and neuronal cell differentiation similar to the induction seen in human ganglioneuroblastomas. Thus, trkA expression in HTLA230 cells is sufficient to generate a functional NGF receptor complex that leads to growth-arrested and differentiated NB cells in vitro and in vivo in the presence of NGF. Hence, NGF may play a crucial role in NB cell differentiation and regression in vivo.

Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA.

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

A metastatic neuroblastoma model in SCID mice.

Neuroblastoma (NB), a neural crest derived tumor in children, shows a characteristic pattern of dissemination that includes adrenal glands, local lymph nodes, bone, liver, skin, and bone marrow. We have reconstructed a similar metastatic pattern in SCID mice following tail vein injection of human NB cells. HTLA230, an NB cell line isolated from a patient with advanced disease, and its NGF receptor (trkA) expressing derivative (18–10) cells, consistently disseminated to the liver, the adrenal gland, and the bone marrow, but not the lungs. Metastases in the different organs showed a characteristic hemorrhagic histopathology, and tumors in the bone marrow presented as syncytia‐like cell aggregates, typically seen in patients. Cell lines reestablished from 18–10 derived liver and bone marrow metastases maintained their cellular morphology, growth behavior, N‐myc overexpression, trkA expression, and functionally responded to NGF treatment, leading to growth arrest and neurite outgrowth. Hence, circulating human NB cells in SCID mice show a similar organ‐specific metastatic potential as seen in patients, independent of trkA expression.

Establishment of a rhabdoid tumor cell line with a specific chromosomal abnormality, 46,XY,t(11;22)(p15.5;q11.23).

The malignant rhabdoid tumor is a rare, poorly understood tumor which occurs primarily in children. The kidney is a frequent primary site of origin, but the tumor has arisen in other mesodermally derived tissues as well. Controversy exists regarding the embryonic origin of the rhabdoid tumor and recent histopathologic studies suggest that it may be of neuroepithelial origin. Our immunohistochemical and electron micrographic studies support this theory. No consistent chromosome abnormalities have been reported in this tumor and no cell lines are available for study. We have established and characterized the first rhabdoid tumor cell line. It possesses a specific chromosomal abnormality, 46,XY,t(11;22)(p15.5;q11.23). The translocation may provide an important clue to the pathogenesis of the tumor as well as an opportunity for further study of the involved chromosome regions.

The RET and TRKA pathways collaborate to regulate neuroblastoma differentiation

Neuroblastoma (NB) is a childhood cancer that arises in the adrenal gland and often shows differentiated neuronal and glial elements. The RET receptor signal pathway is functional in most NB, while loss of nerve growth factor (NGF) receptor (trkA) gene expression correlates with an aggressive phenotype. Thus, we hypothesized that the RET and TRKA signal pathways collaborate to instruct NB differentiation, reminiscent of normal neuronal maturation. Here, we demonstrate that activation of the RET receptor by glial cell line-derived neurotrophic factor (GDNF) increases expression of the RET receptor complex in a panel of malignant human NB cell lines, indicative of a positive feedback mechanism. GDNF also induces growth cessation concomitant with an arrest of cells in the G0/G1 phase of the cell cycle. Furthermore, GDNF synergizes with ciliary neurotrophic factor (CNTF) to enhance TRKA receptor expression, thereby strengthening the NGF-mediated differentiation signal. Differentiated NB cells downregulate expression of the amplified N-myc gene, concurrent with the arrest of cell proliferation, while expressing neuron-specific markers (i.e., SCG10). Interestingly, maintenance of differentiated NB cells in culture is independent of the trophic activity of GDNF, but depends on TRKA signaling, thereby re-enacting the differentiation of normal sympathoadrenal (SA) progenitor cells.

Expression of the functional cone phototransduction cascade in retinoblastoma.

Retinoblastoma is a malignant intraocular tumor that primarily affects small children. These tumors are primitive neuroectodermal malignancies, however some of them show morphologic evidence of differentiation into photoreceptors. Phototransduction cascades are a series of biochemical reactions that convert a photon of light into a neural impulse in rods and cones. The components of these cascades are uniquely expressed in photoreceptors and, although functionally similar, distinct components of these cascades are expressed in rods and cones. Using HPLC anion exchange chromatography, Western blot analysis, and specific monoclonal and polyclonal antibodies, we found that the cone but not the rod cGMP phosphodiesterase is functionally expressed in all six primary retinoblastomas examined and in three continuous retinoblastoma cell lines. Morphologic evidence of differentiation did not correlate with the expression of the enzyme. Furthermore, GTP analogues could activate the phosphodiesterase activity suggesting that an intact phototransduction cascade is present in the tumors. The presence of the cone phototransduction cascade in retinoblastoma confirms that this tumor has biochemically differentiated along the cone cell lineage.

In vitro and in vivo antitumor activity of liposomal Fenretinide targeted to human neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid Fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2‐targeted immunoliposomes (anti‐GD2‐SIL‐HPR). We investigated the antitumor activity of anti‐GD2‐SIL‐HPR against NB cells, both in vitro and in vivo. Anti‐GD2‐SIL showed specific, competitive binding to and uptake by, various NB cell lines. In in vitro cytotoxicity studies, NB cells, incubated with 30 μM HPR entrapped in anti‐GD2‐immunoliposomes, showed a significant reduction in cellular growth compared to free HPR, HPR entrapped in Ab‐free liposomes or anti‐GD2 empty liposomes. In an in vivo NB metastatic model, we demonstrated that anti‐GD2‐SIL‐HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. Similar, but significantly less potent, antitumor effect was observed also in mice treated with anti‐GD2 immunoliposomes without HPR (anti‐GD2‐SIL‐blank) or anti‐GD2 MAb alone (p = 0.0297 and p = 0.0294, respectively, vs. anti‐GD2‐SIL‐HPR). Moreover, our results clearly demonstrated that although anti‐GD2 MAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only after treatment with anti‐GD2‐SIL‐HPR (p < 0.0001). Anti‐GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Regulation of NGF responsiveness in human neuroblastoma.

Functional nerve growth factor (NGF) responsiveness was investigated in human neuroblastoma (NB) cell lines in vitro and retinoic acid (RA) was found to transcriptionally enhance expression of the trkA, but not the trkB gene in GOTO cells, while the reverse was found in HTLA230 NB cells. Ciliary neurotrophic factor (CNTF) specifically induced trkA gene transcription in both cell lines. Transcriptional activation of the trkA gene increased total trkA protein and surface bound receptor, which was tyrosine phosphorylated upon NGF stimulation inducing immediate early response gene transcription (i.e. c-fos, Egr-1). Newly synthesized trkA protein had a molecular weight of 110 kDa and was post-translationally modified. Rapid down regulation of the receptor protein occurred upon NGF stimulation, despite the presence of high levels of trkA mRNA, due to an increased rate of receptor degradation. Transient DNA synthesis and cell proliferation upon NGF treatment occurred in GOTO cells with elevated trkA expression. In contrast, NGF induced neuronal differentiation in HTLA230 cells expressing the endogenous trkA receptor gene, despite the lack of p75 expression. Hence, transcriptional activation of trkA gene expression can be achieved by different signal pathways in human NB cells, but NGF can act either as mitogen or inducer of cell differentiation, depending on the tumor from which cells are derived.

Osmotic Swelling Induces p75 Neurotrophin Receptor (p75NTR) Expression via Nitric Oxide

Brain injuries by physical trauma, epileptic seizures, or microbial infection upset the osmotic homeostasis resulting in cell swelling (cerebral edema), inflammation, and apoptosis. Expression of the neurotrophin receptor p75NTR is increased in the injured tissue and axon regeneration is repressed by the Nogo receptor using p75NTR as the signal transducer. Hence, p75NTR seems central to the injury response and we wished to determine the signals that regulate its expression. Here, we demonstrate that tonicity mediated cell swelling rapidly activates transcription of the endogenous p75NTR gene and of a p75NTR promoter-reporter gene in various cell types. Transcription activation is independent of de novo protein synthesis and requires the activities of phospholipase C, protein kinase C, and nitric-oxide synthase. Hence, p75NTR is a nitric oxide effector gene regulated by osmotic swelling, thereby providing a strategy for therapeutic intervention to modulate p75NTR functions following injury.