Faculty Bibliography

The IL-2 dependent murine cytotoxic T cell line CTLL-2 undergoes programmed cell death when deprived of its specific cytokine. We analyzed the expression of cell cycle related genes after IL-2 deprivation. Here we show that a generalized decrease and re elevation of the levels of mRNA takes place as part of the apoptotic program. The levels of several mRNAs encoding cell cycle functions, including cyclin D2, cyclin D3, cyclin B1, c-myc and max all declined at 1.5-3 h following IL-2 deprivation. Notably, the maxmRNA, which was shown to be expressed in proliferating, growth arrested and differentiated cells, is down regulated with the same kinetics as the other mRNAs. Surprisingly, the mRNAs whose levels declined at 1.5-3 h rose again at 10-14 h, a time which closely followed the time of the first detection of apoptotic DNA degradation, at 8 h, but which precedes actual loss of viability, at 14 h, as measured by trypan blue exclusion. Of all analyzed genes only the expression of the S-phase specific histone H4 gene resists the initial decrease and declines gradually over the course of cell death. Measurement of c-Myc protein synthesis at a late stage of the apoptotic program revealed that the accumulated reinduced mRNA is not translated into protein. Because transcriptional regulation has been shown to be dependent on the chromatin structure, the reinduction may be triggered by relaxation of the chromatin caused by alterations in the chromatin structure of apoptotic cells

We have analysed the molecular basis for the function of the C/EBP alpha transactivation domain. We have previously found that the three C/EBP alpha transactivation elements (TEs) synergistically activate transcription in mammalian cells. We now report that two of these elements, TE-I and -II, co-operatively mediate in vitro binding of C/EBP alpha to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP alpha coincide, and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and -II in both yeast and mammalian cells. Our data demonstrate a biochemical basis for the modular buildup of transactivation domains, and indicate that this modularity is conserved in eukaryote evolution. We also show that the same amino acid motifs in a cellular activator can co-operate to mediate contacts between the activator and two distinct basal transcription factors. These results suggest that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related, and support the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process

We have examined the effects of NGF on components of the PC12 cell cycle machinery. We show that NGF represses over 6-8 d the levels of specific cdk kinase proteins and the G2-M phase specific cyclin B1 and the S phase marker PCNA as well as the level of phosphorylation of the retinoblastoma (Rb) protein. All of these changes may provide a basis for a NGF block to cell cycling. Unexpectedly, the G1 phase-specific cyclin D1 was dramatically increased by inducers of differentiation (NGF and FGF), but not by inducers of proliferation (EGF and insulin). Although the levels of cyclin D1/cdk2 and cyclin D1/cdk4 complexes increased following NGF treatment, as did cyclin D1/Rb complexes, the associated kinase activities declined, indicating that NGF also induces an inhibitor of cdk kinase activity. In agreement, NGF induced the cdk inhibitory protein, p21, which was found in cyclin D1/cdk kinase complexes after NGF treatment. We show that vector over expression of cyclin D1 in PC12 is sufficient on its own to arrest the cells in G1 phase and inhibit expression of PCNA. These results indicate that NGF induction of cyclin D1 and inactivation of cdk kinases, the latter possibly by increase of p21, play a central role in the NGF block of PC12 cell cycling

Heterodimerization of Max with the nuclear oncoprotein Myc and the differentiation-associated proteins Mad and Mxi1 enables these factors to bind E-box sites in DNA and control genes implicated in cell proliferation and differentiation. We show that in the PC12 pheochromocytoma tumor cell line, functional Max protein is not expressed because of the synthesis of a mutant max transcript. This transcript encodes a protein incapable of homo- or heterodimerization. Furthermore, the mutant Max protein, unlike wild-type Max, is incapable of repressing transcription from an E-box element. Synthesis of mutant max transcripts appears to be due to a homozygous chromosomal alteration within the max gene. Reintroduction of max into PC12 cells results in repression of E-box-dependent transcription and a reduction in growth rate, which may explain the loss of Max expression either during the growth of the pheochromocytoma or in subsequent passage of the PC12 cell line in vitro. Finally, the ability of these cells to divide, differentiate, and apoptose in the absence of Max demonstrates for the first time that these processes can occur via Max- and possibly Myc-independent mechanisms

Using recombinant baculovirus expressed p53 and Raf proteins, we show that activated Raf-1 kinase can phosphorylate mouse p53 in vitro. We also show that co-expression of vRaf and p53 in NIH3T3 fibroblasts, potentiates the ability of p53 to transactivate a minimal promoter with a p53 cognate DNA binding site. A dominant negative mutant of Raf inhibits the transactivation function of p53 in NIH3T3 fibroblasts. Incubation of Raf-1 kinase with a series of p53 derived synthetic peptides maps the Raf-1 phosphorylation sites to the 27 amino terminal residue region of p53 which coincides with the transactivation domain. Phosphorylation occurs on serines which are phosphorylated in vivo. Our results suggest that the transactivation function of p53 can be regulated by a signaling cascade involving Raf

The D-type cyclins are growth factor-regulated delayed early functions which peak at the G1/S transition, are thought to regulate entry into S phase and have been implicated in tumorigenesis. Here, we show that cyclin D2 can co-operate with Ha-Ras to impose a novel transformed state on rat embryo fibroblasts (REF). While clonal cyclin D2/Ha-Ras REF transformants exhibit a characteristic transformed phenotype in high serum, in low serum they arrest cell proliferation and display profound morphological and cytological changes indicating loss of control of cell mass and deregulation of the G1/S transition. Notably, in low serum, despite re-establishment of actin cables and arrest of proliferation, cell mass continues to increase, creating giant cells up to 10 x normal size. Also, during low-serum culture the cells make a very gradual but progressive entry into S phase, reaching a 2.4N DNA content after 6 days. PCNA is expressed and 2N and 4N cells are largely absent, and thus the cells undergo a novel S phase arrest. While transfer to low serum induced the retinoblastoma protein to enter its dephosphorylated state, and cyclin A, cyclin B and cdc2 levels to decrease, all as normal, cyclin E, cdk4, cdk2 and the exogenous cyclin D2 persisted at high levels. These results indicate that cyclin D2 and Ha-Ras can transform cells when mitogenic signals from growth factors are provided. However, in low serum, co-operation of cyclin D2 and Ha-Ras provides only a subset of the progression signals and these are sufficient for G1-related cell mass increase and S phase entry, but are insufficient for full cell cycling

Myc is a nuclear phosphoprotein which controls cellular proliferation, most likely by regulating gene activity. The finding that the neuronal model cell line PC12 lacks the Myc DNA binding partner, the Max protein, and the demonstration that Myc is a repressor of gene activity as well as a transactivator, lead to models for Myc action in regulating cell growth