Regulation of cell lineage specification by the retinoblastoma tumor suppressor.

Early studies of the retinoblastoma gene (RB) have uncovered its critical role as a regulator of the G(1)/S cell cycle phase progression. Surprisingly, genetic approaches in mammals and nematodes have also shown RB controls cell lineage specification and aspects of differentiation. The RB gene product accomplishes this by diverse mechanisms such as by interacting with tissue-specific transcription factors, enhancing RNA interference, and modifying chromatin structure. We review recent studies uncovering novel mechanisms by which RB works in several cell lineages and we provide perspectives on how these new findings might relate to RB tumor suppression.

Persistent expression of cyclin D1 disrupts normal photoreceptor differentiation and retina development.

The differentiation of neuronal cells in the developing mammalian retina is closely coupled to cell cycle arrest and proceeds in a highly organized manner. Cyclin D1, which regulates cell proliferation in many cells, also drives the proliferation of photoreceptor progenitors. In the mouse retina, cyclin D1 protein normally decreases as photoreceptors mature. To study the importance of the down-regulation of cyclin D1 during photoreceptor development, we generated a transgenic mouse in which cyclin D1 was persistently expressed in developing photoreceptor cells. We observed numerous abnormalities in both photoreceptors and other nonphotoreceptor cells in the retina of these transgenic mice. In particular, we observed delayed opsin expression in developing photoreceptors and alterations in their number and morphology in the mature retina. These alterations were accompanied by disorganization of the inner nuclear and plexiform layers. The expression of cyclin D1 caused excess photoreceptor cell proliferation and apoptosis. Loss of the p53 tumor suppressor gene decreased cyclin D1-induced apoptosis and led to microscopic hyperplasia in the retina. These findings are distinct from other mouse models in which the retinoblastoma gene pathway is disrupted and suggest that the IRBP-cyclin D1 mouse model may recapitulate an early step in the development of retinoblastoma.

The proliferative and apoptotic activities of E2F1 in the mouse retina.

The E2F1 transcription factor controls cell proliferation and apoptosis. E2F1 activity is negatively regulated by the retinoblastoma (RB) protein. To study how inactivation of Rb and dysregulated E2F1 affects the developing retina, we analysed wild-type and Rb(-/-) embryonic retinas and retinal transplants and we established transgenic mice expressing human E2F1 in retinal photoreceptor cells under the regulation of the IRBP promoter (TgIRBPE2F1). A marked increase in cell proliferation and apoptosis was observed in the retinas of Rb(-/-) mice and TgIRBPE2F1 transgenic mice. In the transgenic mice, photoreceptor cells formed rosette-like arrangements at postnatal days 9 through 28. Complete loss of photoreceptors followed in the TgIRBPE2F1 mice but not in the Rb(-/-) retinal transplants. Both RB-deficient and E2F1-overexpressing photoreceptor cells expressed rhodopsin, a marker of terminal differentiation. Loss of p53 partially reduced the apoptosis and resulted in transient hyperplasia of multiple cell types in the TgIRBPE2F1 retinas at postnatal day 6. Our findings support the concept that cross-talk occurs between different retinal cell types and that multiple genetic pathways must become dysregulated for the full oncogenic transformation of neuronal retinal cells.

Cytogenetics and the biologic basis of sarcomas.

In this past year, a large number of reports have described cytogenetic and biologic studies of sarcomas. The cytogenetic studies provide further evidence that a growing number of sarcomas seem to be defined by consistent chromosomal abnormalities that can be detected using a variety of molecular genetic tests. However, in addition to these specific abnormalities, many sarcomas have other extremely complex genetic changes. This complexity has made it quite difficult to understand the importance of any single abnormality. Laboratory studies complementing these genetic studies have provided further understanding of sarcoma cellular and molecular biology. Importantly, both types of studies have had significant impact in the clinic in the form of more objective diagnostic tests, potential novel prognostic markers, and even new therapeutic strategies. Together, these papers highlight how genetic studies may offer tremendous insight into sarcoma biology. However, they also highlight some limitations of these approaches as well. Novel experimental approaches may be required to facilitate the continued progress in this field toward the development of better therapeutic strategies.

Cloning and characterization of a novel Kruppel-associated box family transcriptional repressor that interacts with the retinoblastoma gene product, RB.

The retinoblastoma gene product, RB, seems to function as a key tumor suppressor by repressing the expression of genes activated by members of the E2F family of transcription factors. In order to accomplish this, RB has been proposed to interact with a transcriptional repressor. However, no genuine transcriptional repressors have been identified by virtue of interaction with RB. By using the yeast two-hybrid system, we have identified a novel member of a known family of transcriptional repressors that contain zinc fingers of the Kruppel type and a portable transcriptional repressor motif known as the Kruppel-associated box (KRAB). The mouse and human forms of the novel RB-associated KRAB protein (RBaK) are widely expressed. The amino acid motif that links the KRAB domain and zinc fingers appears to be required for interaction with RB in vitro. Human RBaK ectopically expressed in fibroblasts is an 80-kDa protein that is localized to the nucleus. The expression of either RB or RBaK in 10T1/2 fibroblasts represses the activation of an E2F-dependent promoter and decreases DNA synthesis to a similar degree. However, a mutant form of RBaK that cannot interact with RB in vitro is unable to prevent DNA synthesis. We present a model in which RB physically interacts with the novel transcriptional repressor RBaK to repress E2F-dependent genes and prevent DNA synthesis.

Combination chemotherapy using vinblastine and methotrexate for the treatment of progressive desmoid tumor in children.

PURPOSE: We report the treatment of 10 children for progressive desmoid tumor not amenable to standard surgical or radiation therapy with the use of vinblastine (VBL) and methotrexate (MTX). PATIENTS AND METHODS: Ten patients aged 6.4 to 18 years with primary (two patients) or recurrent (eight patients) desmoid tumor were treated with VBL and MTX for 2 to 35 months. Patients with recurrent tumors had been previously treated with surgical resection with (two patients) or without (five patients) radiation therapy or with radiation therapy alone (one patient). No patient had previously received cytotoxic chemotherapy. The tumor response was assessed at routine intervals by physical examination and magnetic resonance imaging (MRI). RESULTS: Five patients had clinical evidence of response to therapy with complete resolution (three patients) or partial resolution (two patients) of physical examination and radiographic abnormalities. Three patients had stable disease during 10 to 35 months of treatment. Two of these patients had progressive disease 9 and 37 months after treatment stopped; one patient had no progression 16 months after therapy. Two additional patients with stable disease had chemotherapy discontinued after 2 and 3 months. Common side effects included mild alopecia and myelosuppression and moderate nausea and vomiting. In patients with responding tumors, MRI showed decreased tumor size and, in two patients, changes consistent with fibrosis and decreased cellularity of the tumor. CONCLUSION: Combination chemotherapy with VBL and MTX appears to control desmoid tumor without significant acute or long-term morbidity in most children. This may allow for further growth and development in these patients, which may decrease the morbidity of subsequent definitive therapy.

Cytogenetics and the biological basis of sarcomas.

Recently, much research has been directed toward gaining a better understanding of sarcoma biology. To accomplish this goal, researchers have focused on characterizing the cytogenetic abnormalities that are detectable by routine karyotyping. With the use of widely-available molecular biologic tools, new information on the genetic abnormalities of sarcomas is rapidly emerging. In addition, physicians are beginning to successfully apply cytogenetic and molecular biologic findings to clinical settings in the form of molecular diagnostic and prognostic tests. Moreover, detailed study of these genetic abnormalities is leading to a better understanding of the molecular pathology of sarcomas, which may eventually lead to better therapy. In this paper, we will review the important new findings on genetic abnormalities in sarcomas, clinical applications of cytogenetic studies, and insight into the biology of sarcomas.

Asparaginase-associated lipid abnormalities in children with acute lymphoblastic leukemia.

To further elucidate the incidence and potential mechanism of asparaginase-associated lipid abnormalities in children with acute lymphoblastic leukemia (ALL), we serially obtained fasting lipid and lipoprotein studies on 38 of the 43 consecutively diagnosed children with ALL before, during, and after asparaginase therapy. We also evaluated a second population of 30 long-term survivors of childhood ALL; a fasting lipid and lipoprotein profile was obtained once at study entry. The mean peak triglyceride level during asparaginase of 465 mg/dL (standard deviation [SD] 492) was significantly higher (P = .003) than the level of 108 mg/dL (SD 46) before the initiation of asparaginase therapy. Sixty-seven percent of the newly diagnosed patients had fasting triglyceride levels greater than 200 mg/dL during asparaginase therapy; 15 patients (42%) had levels greater than 400 mg/ dL, 7 with levels greater than 1,000 mg/dL. The incidence of hypertriglyceridemia did not vary by type of asparaginase or risk status of ALL (defined by white blood cell count and age). None of the 7 patients with triglyceride levels greater than 1,000 mg/dL developed pancreatitis. In contrast, 4 of the 13 patients without triglyceride elevation developed pancreatitis; 3 of the 4 patients had fasting studies at the height of their abdominal pain. Nuclear magnetic resonance analysis of lipid subclasses showed a significant increase in the smaller, denser forms of very low density lipoprotein (VLDL) and negligible chylomicron fraction in a subset of patients with marked triglyceride elevation. Lipoprotein lipase activity was consistently above normative values for all levels of triglyceride and could not be explained by obesity or hyperglycemia. Apolipoprotein B(100) levels increased during asparaginase therapy, although the mechanism of this remains unclear. LDL reciprocally decreased with increased VLDL during asparaginase therapy. After asparaginase therapy, triglyceride levels (mean, 73 mg/dL [SD 33]) were significantly lower than levels obtained during asparaginase therapy. Triglyceride levels for survivors did not differ from the normal range or postasparaginase levels in the newly diagnosed patients. These data show a striking temporal association between asparaginase therapy and hypertriglyceridemia. Changes in cholesterol, in contrast, were not temporally related to asparaginase treatment. Cholesterol levels were elevated (>200 mg/dL) in 20% of the patients after asparaginase, which may be due to continued treatment with corticosteroids. The mean cholesterol level of long-term survivors of 177 mg/dL was significantly higher than the norm (P = .045). High-density lipoprotein (HDL) levels were significantly lower than normal at all time periods and for both populations; 25% of survivors had HDL levels less than 35 mg/dL. We conclude that modifications in asparaginase therapy are not necessary. In cases of triglyceride elevation greater than 2,000 mg/dL when the risk of pancreatitis is increased, close clinical monitoring is imperative. Larger studies are needed to determine the incidence of dyslipidemia in long-term survivors of ALL as well as the relationship between lipid abnormalities and other late effects of treatment, notably obesity and cardiomyopathies.

Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation.

It was recently demonstrated that ectopic expression of cyclin D1 inhibits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. Chem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cyclin D1 inhibits muscle gene expression without affecting MyoD DNA binding activity. Ectopic expression of cyclin D1 inhibits muscle gene activation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absent. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediated inhibition of myogenesis was due to hyperphosphorylation of pRB. In pRB-deficient fibroblasts, the ability of MyoD to activate the expression of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expression. The negative effects of cyclins A or E on muscle gene expression are, however, reversed by the presence of a mutated form of pRB which cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit muscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G1 cyclin-cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphosphorylation and one that is independent of pRB hyperphosphorylation.

Genes in the RB pathway and their knockout in mice.

The retinoblastoma susceptibility gene (RB), the first identified human tumor suppressor gene, has been shown to be directly involved in the genesis of a variety of human cancers. RB is actually one of a family of three closely related genes including p107 and p130. Many elegant biochemical studies have demonstrated that RB is a critical component of the cell cycle regulatory machinery and have characterized the downstream effectors which the RB gene product regulates. More recent advances have demonstrated that the function of RB and RB-related genes is positively and negatively regulated by an intricate network of cell cycle regulatory proteins, some of which have also been implicated as tumor suppressor genes. Despite the detailed understanding of these biochemical and genetic pathways, the full function of genes in the RB pathway in the context of a whole organism is only now being addressed. Using gene knockout technology, it is now known that RB, and RB-related proteins p107 and p130, have important functions during early mouse development. Furthermore, despite its ubiquitous expression, RB has tissue- and cell-type specific effects which account for its function as a tumor suppressor but may also be independent of its role as a cell cycle regulator. Analysis of mice lacking regulatory genes upstream of RB and effector genes downstream of RB have confirmed that other genes in this pathway have tissue-specific effects on development and tumor susceptibility in mice.

Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD.

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Inhibition of myogenic differentiation in proliferating myoblasts by cyclin D1-dependent kinase.

Although the myogenic regulator MyoD is expressed in proliferating myoblasts, differentiation of these cells is limited to the G0 phase of the cell cycle. Forced expression of cyclin D1, but not cyclins A, B, or E, inhibited the ability of MyoD to transactivate muscle-specific genes and correlated with phosphorylation of MyoD. Transfection of myoblasts with cyclin-dependent kinase (Cdk) inhibitors p21 and p16 augmented muscle-specific gene expression in cells maintained in high concentrations of serum, suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating cells.

Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal.

Skeletal muscle differentiation entails the coupling of muscle-specific gene expression to terminal withdrawal from the cell cycle. Several models have recently been proposed which attempt to explain how regulated expression and function of myogenic transcription factors ensures that proliferation and differentiation of skeletal muscle cells are mutually exclusive processes.

Melphalan-induced toxicity in nude mice following pretreatment with buthionine sulfoximine.

Melphalan-induced toxicity in nude mice following pretreatment with a regimen of L-buthionine sulfoximine (BSO), previously shown to enhance the activity of this alkylating agent against rhabdomyosarcoma and glioma xenografts, was examined. Mice were pretreated with i.p. BSO (2.5 mmol/kg x 7 doses at 12-h intervals plus concomitant availability of a 20-mM solution in the drinking water) or vehicle prior to a single i.p. injection of melphalan (35.65 mg/m2). As compared with control animals who received no BSO pretreatment, mice pretreated with BSO lost weight prior to therapy with melphalan (6.9% weight loss vs 0.3% weight gain; P less than 0.005) and showed a greater mean nadir weight loss after melphalan (3.8% vs. 2.1%; P = 0.049). Treatment with melphalan was associated with histologic evidence of reversible gastrointestinal toxicity, reversible myelosuppression, and histologic evidence of acute renal tubular necrosis, with no differences being observed between mice that had been pretreated with BSO and those that had been pretreated with vehicle. No evidence of cardiac, hepatic, or skeletal muscle toxicity was found in melphalan-treated animals. These results suggest that treatment of nude mice with melphalan following BSO-mediated depletion of glutathione does not result in enhanced organ toxicity despite an increase in the antineoplastic activity of this alkylating agent.

Phenotypic analysis of four human medulloblastoma cell lines and transplantable xenografts.

An extensive panel of monoclonal antibodies (MAb) and monospecific antisera reactive against neuroectodermal-, neuronal-, glial-, and lymphoid-associated antigens, extracellular matrix, HLA, and cell-surface receptors was used to characterize the phenotype of four continuous, karyotypically distinct medulloblastoma cell lines and transplantable xenografts. All four cell lines demonstrated significant reactivity with anti-neuroectodermal-associated MAb. No apparent pattern of reactivity with anti-lymphoid MAb was seen; notably, there was a uniform absence of detectable Thy-1. Review of the complete antibody reactivity profile revealed a dichotomy between lines TE-671 and Daoy and lines D283 Med and D341 Med, which have been previously shown to express neurofilament protein in culture and xenografts, and to exhibit neuroblastic morphological features in biopsy and xenograft tissue sections. TE-671 and Daoy reacted with the MAb directed against tenascin, epidermal growth factor (EGF) receptor, HLA-A,B epitopes, beta 2-microglobulin and 5/8 of the glioma-associated antigens, but did not react with the anti-neurofilament protein (NFP) MAb. D283 Med and D341 Med expressed NFP but did not react with MAb against tenascin, EGF receptor, HLA-A,B epitopes, beta 2-microglobulin or 6/8 and 7/8 (respectively) of the glioma-associated antigens. The observed phenotypic differences provide a conceptual framework for investigating basic differences in the biological behavior of medulloblastoma. Moreover, the subdivisions can be evaluated for prospective value in tissue diagnosis, cerebrospinal fluid cytology and antibody-mediated imaging and therapy.

Buthionine sulfoximine-mediated depletion of glutathione in intracranial human glioma-derived xenografts.

D-54 MG, a human glioma-derived continuous cell line growing as subcutaneous or intracranial xenografts in athymic mice, was found to be sensitive to the effects of D,L-buthionine-(SR)-sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase. Intraperitoneal administration of one dose of buthionine sulfoximine (BSO, 5 mmol/kg) resulted in depletion of total intracellular glutathione to 57 and 47% of control 12 hr, and 73 and 23% of control 24 hr, after BSO in subcutaneous and intracranial xenografts respectively. Concurrent measurement of total glutathione in the contralateral (non-tumor-containing) cerebral hemisphere in mice bearing intracranial D-54 xenografts demonstrated insignificant depletion of glutathione. Multiple doses of BSO, at 12-hr intervals, resulted in further depletion to 27% (s.c.) and 16.5% (i.c.) of control 12 hr following the final dose of BSO. Quantitative analysis of BSO delivery to xenograft and contralateral brain tissue revealed transfer constants, K1, of 15.8-24.1 x 10(-3) and 2.4 x 10(-3) ml.g-1.min-1 for xenograft and "normal" brain respectively. This highly selective depletion of glutathione in neoplastic tissue versus surrounding non-neoplastic host tissue may have therapeutic implications for the rational use of chemotherapeutic and radiotherapeutic intervention.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Experimental chemotherapy of human medulloblastoma cell lines and transplantable xenografts with bifunctional alkylating agents.

A series of bifunctional alkylators were tested against the genotypically and phenotypically heterogeneous continuous human medulloblastoma cell lines, TE-671, Daoy, and D283 Med in vitro and against TE-671 and Daoy growing as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (in vivo), and busulfan (in vivo). Melphalan and phenylketocyclophosphamide were the most active agents in vitro with drug doses at which there is a 90% reduction in the number of colonies in comparison to controls of 2.13, 5.29, and 4.72 microM for melphalan and 4.60, 5.01, and 4.34 microM for phenylketocyclophosphamide against TE-671, D283 Med, and Daoy, respectively. Melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, and thiotepa produced significant growth delays against s.c. TE-671 and Daoy xenografts, while no activity could be demonstrated for 1,3-bis(2-chloroethyl)-1-nitrosourea or busulfan. Melphalan, cyclophosphamide, iphosphamide, and thiotepa also produced significant increases in median survival in mice bearing intracranial TE-671 and Daoy xenografts. These results extend our previous studies demonstrating the antitumor activity of nitrogen and phosphoramide mustard-based bifunctional alkylating agents in the treatment of human medulloblastoma continuous cell lines and transplantable xenografts, and support the continued use of these agents in clinical trials.

Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in a human medulloblastoma xenograft in athymic mice.

The effect and therapeutic consequences of buthionine-(SR)-sulfoximine (BSO)-mediated depletion of glutathione in the human medulloblastoma-derived cell line, TE-671, growing as s.c. xenografts in athymic nude mice were examined. The glutathione content of the s.c. xenografts was 1.11 +/- 0.15 mumol/g (7.79 +/- 1.61 nmol/mg of protein). Administration i.p. to tumor-bearing mice of D,L-BSO (two doses at 12-h intervals; 5 mmol/kg) depleted the glutathione content of the xenografts to 25.7% of control. Administration of a 30 mM solution of L-BSO in drinking water for 96 h depleted the glutathione content to 17.4% of control. Depletion of glutathione with these regimens resulted in a significant increase in the s.c. tumor growth delay over that produced by melphalan alone: 17.2 days versus 12.6 days for D,L-BSO (i.p.) plus melphalan versus melphalan and 22.9 days versus 16.6 days for L-BSO (p.o.) plus melphalan versus melphalan. These studies demonstrate the increased cytotoxicity of melphalan resulting from BSO-mediated depletion of glutathione in human medulloblastoma and support further efforts to modulate the chemosensitivity and radiosensitivity of this tumor by modulation of glutathione.