p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells.

This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.

The CXCR4 inhibitor BL-8040 induces the apoptosis of AML blasts by downregulating ERK, BCL-2, MCL-1 and cyclin-D1 via altered miR-15a/16-1 expression.

CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.

Reduced levels of alpha 1 (I) collagen mRNA in cells immortalized by mutant p53 or transformed by ras.

Genes whose expression patterns are altered in a cell line immortalized by mutant p53 were isolated by differential screening of a cDNA library. Levels of alpha 1 (I) collagen mRNA were reduced in the majority of immortalized cell lines which greatly overproduced the transfected mutant p53. This may reflect a co-selection during the establishment of the cell lines, rather than a direct effect of p53 on alpha 1 (I) collagen gene expression. On the other hand, a more direct relationship could be demonstrated between the expression of activated ras and a reduction in alpha 1 (I) collagen mRNA. Such reduction could partially account for the effects of ras on cell shape and cell proliferation.

Linear dimeric interleukin-2 obtained by the use of a defective herpes simplex viral vector: conformation-activity relationship.

An interleukin-2 dimer, produced enzymatically by a nerve-derived transglutaminase in vitro, is cytotoxic to oligodendrocytes, unlike the immune-derived monomeric interleukin-2. The object of this study was to establish a way to produce a dimer of interleukin-2 in quantities, by means of genetic engineering, and to confirm that the structure of the resulting molecule is critical for its function. A defective herpes simplex virus vector was utilized for overproduction of a dimeric interleukin-2. The resulting linear dimer, which is a translational product, differs from the enzymatically produced dimer, which is a posttranslational modification of interleukin-2. The linear dimer, while retaining the known interleukin-2 activity of monomeric interleukin-2 with respect to mitogenicity on T cells, was not cytotoxic to oligodendrocytes. This finding suggests that the lack of cytotoxicity of the linear dimeric interleukin-2 is not caused by a loss of activity during its preparation but is related to its conformational structure, which evidently does not meet the requirements for cytotoxicity. This study opens the way to the design at the transcriptional level of modified proteins and their efficient production, provided that the new transcript encodes for the desired modification in the protein at the appropriate sites.

The CXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of the bone marrow after transplantation.

Cytopenia represents a significant complication after chemotherapy, irradiation before bone marrow (BM) transplantation or as a therapy for cancer. The mechanisms that determine the pace of BM recovery are not fully understood. During the recovery phase after chemotherapy or irradiation, the signals for retention of white blood cells within the BM increase significantly. This leads to a delay in the release of WBC, which can be overcome by targeting the CXCR4 axis with the antagonist 4F-benzoyl-TN14003 (T140). The delay in the release of WBC is also accompanied by suppression in the production of progenitor cells and mature cells by the BM stroma. Administration of T140 to mice transplanted with BM cells stimulates the production of all types of progenitors and mature cells, and increases the exit of mature cells to the periphery. Moreover, addition of T140, but not AMD3100, to BM stromal cultures stimulates the production of mature cells and progenitors from all lineages. The unique ability of the CXCR4 antagonist, T140 to stimulate the production and exit of WBC cells may be used as a novel therapeutic approach to overcome cytopenia associated with treatments for cancer and BM transplantation.

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1,700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1,500, 1,850, 1,850 and 2,500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

Interleukin-2 transcripts in human and rodent brains: possible expression by astrocytes.

Cytokines have been suggested to be involved in the cross talk between the immune and the nervous systems, under normal and pathological conditions. For example, the cytokine interleukin-2 was suggested to be involved in response to CNS trauma and spontaneous regeneration. Here, we examined whether mammalian CNS has an intrinsic potential to produce interleukin-2 and, if so, what its cellular origin is. mRNA sequences encoding for interleukin-2 were detected in brains of humans and rodents. Northern blot analysis revealed the presence of several interleukin-2 transcripts of different sizes in the brain, all recognized by lymphocyte-derived interleukin-2 cDNA probes. One of the transcripts, a high molecular weight form of approximately 5 kb, appeared to be unique to the brain. Reverse transcription and amplification by PCR of human fetal brain mRNA revealed one cDNA product that, upon sequence analysis, showed a high degree of homology with the human lymphocyte-derived interleukin-2 coding sequence. To identify the possible cellular source of the interleukin-2 transcripts within the mammalian brain, we similarly analyzed mRNA of rat brain cells in culture. Northern blot analysis revealed that astrocytes contain transcripts that hybridize with interleukin-2 cDNA probe. These findings point to the astrocytes as a possible source of brain interleukin-2.

Sequence homology within the morbilliviruses.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA extracted from monkey kidney cells infected with canine distemper virus (CDV) was cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing CDV DNA were identified by hybridization to a CDV-specific 32P-labeled cDNA. A cDNA clone containing an insert 1,700 base pairs (CDV 364) has been identified as the reverse transcript of the mRNA coding for the nucleocapsid protein. The size of the mRNA species complementary to this insert is 1,850 nucleotides, as determined by the Northern technique. Hybridization experiments and heteroduplex mapping indicated homology between the central region of the CDV and measles virus nucleocapsid gene. The completion of the nucleotide sequence analysis of the measles virus gene allowed the reconstruction of the entire coding region of the measles virus gene and a comparison with the counterpart sequence of CDV. This comparison delineated three regions: (i) a region of high homology (nucleotides 501 to 1215), in which 77% of the nucleotides and 88% of the encoded amino acids are identical; (ii) a region of moderate homology at the 5' end of the message (nucleotides 1 to 500), in which 59% of the nucleotides and 66% of the encoded amino acids are identical; (iii) a region of little or no homology (nucleotides 1216 to 1625) near the 3' end of the message.

Direct involvement of p53 in programmed cell death of oligodendrocytes.

A covalent dimer of interleukin (IL)-2, produced in vitro by the action of a nerve-derived transglutaminase, has been shown previously to be cytotoxic to mature rat brain oligodendrocytes. Here we report that this cytotoxic effect operates via programmed cell death (apoptosis) and that the p53 tumor suppressor gene is involved directly in the process. The apoptotic death of mature rat brain oligodendrocytes in culture following treatment with dimeric IL-2 was demonstrated by chromatin condensation and internucleosomal DNA fragmentation. The peak of apoptosis was observed 16-24 h after treatment, while the commitment to death was already observed after 3-4 h. An involvement of p53 in this process was indicated by the shift in location of constitutively expressed endogenous p53 from the cytoplasm to the nucleus, as early as 15 min after exposure to dimeric IL-2. Moreover, infection with a recombinant retrovirus encoding a C-terminal p53 miniprotein, shown previously to act as a dominant negative inhibitor of endogenous wild-type p53 activity, protected these cells from apoptosis.

Expression of GTP-dependent and GTP-independent tissue-type transglutaminase in cytokine-treated rat brain astrocytes.

Tissue-type transglutaminases (TGases) were recently shown to exert dual enzymatic activities; they catalyze the posttranslational modification of proteins by transamidation, and they also act as guanosine triphosphatase (GTPase). Here we show that a tissue-type TGase is expressed in rat brain astrocytes in vitro, and is induced by the inflammation-associated cytokines interleukin-1beta and to a lesser extent by tumor necrosis factor-alpha. Induction is accompanied by overexpression and appearance of an additional shorter clone, which does not contain the long 3'-untranslated region and encodes for a novel TGase enzyme whose C terminus lacks a site that affects the enzyme's interaction with guanosine triphosphate (GTP). Expression of two clones revealed that the long form is inhibited noncompetitively by GTP, but the short form significantly less so. The different affinities for GTP may account for the difference in physiological function between these two enzymes.

A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice.

Brain injury induces disruption of the blood-brain barrier, edema, and release of autodestructive factors that produce delayed neuronal damage. NAPSVIPQ (NAP), a femtomolar-acting peptide, is shown to be neuroprotective in a mouse model of closed head injury. NAP injection after injury reduced mortality and facilitated neurobehavioral recovery (P < 0.005). Edema was reduced by 70% in the NAP-treated mice (P < 0.01). Furthermore, in vivo magnetic resonance imaging demonstrated significant brain-tissue recovery in the NAP-treated animals. NAP treatment decreased tumor necrosis factor-alpha levels in the injured brain and was shown to protect pheochromocytoma (PC12 cells) against tumor necrosis factor-alpha-induced toxicity. Thus, NAP provides significant amelioration from the complex array of injuries elicited by head trauma.