Telomere dysfunction alters the chemotherapeutic profile of transformed cells.

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC(-/-)) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.

Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation.

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.

D3: a gene induced during myeloid cell differentiation of Linlo c-Kit+ Sca-1(+) progenitor cells.

In an effort to characterize molecular events contributing to lineage commitment and terminal differentiation of stem/progenitor cells, we have used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and cell lines blocked at two distinct stages of differentiation. The cell lines used were EML, which is representative of normal multipotential primitive progenitors (Sca-1(+), CD34(+), c-Kit+, Thy-1(+)) able to differentiate into erythroid, myeloid, and B-lymphoid cells in vitro, and MPRO, which is a more committed progenitor cell line, with characteristics of promyelocytes able to differentiate into granulocytes. One clone isolated by this approach was expressed in MPRO but not in EML cells and contained sequence identical to the 3' untranslated region of D3, a gene cloned from activated peritoneal macrophages of unknown function. We have observed a novel pattern of D3 gene expression and found that D3 is induced in EML cells under conditions that promote myeloid cell differentiation (interleukin-3 [IL-3], stem cell factor [SCF], and all-trans-retinoic acid [atRA]) starting at 2 days, corresponding to the appearance of promyelocytes. D3 RNA expression reached a maximum after 5 days, corresponding to the appearance of neutrophilic granulocytes and macrophages, and decreased by day 6 with increased numbers of differentiated neutrophils and macrophages in vitro. Induction of D3 RNA in EML was dependent on IL-3 and was not induced in response to SCF or atRA alone or SCF in combination with 15 other hematopoietic growth factors (HGF) tested. Similarly, D3 was not expressed in the normal bone marrow cell (BMC) counterpart of EML cells, Linlo c-Kit+ Sca-1(+) progenitor cells. D3 RNA expression was induced in these cells when cultured for 7 days in IL-3 plus SCF. A comparison of the expression of D3 RNA in cell lines and normal BMC populations demonstrated that D3 is induced during macrophage and granulocyte differentiation and suggests a potential physiological role for D3 in normal myeloid differentiation.

C/EBPepsilon is a myeloid-specific activator of cytokine, chemokine, and macrophage-colony-stimulating factor receptor genes.

C/EBPepsilon is a member of the CCAAT/enhancer binding protein family of basic region/leucine zipper transcriptional activators. The C/EBPepsilon protein is highly conserved between rodents and humans, and its domain structure is very similar to C/EBPalpha. In mice C/EBPepsilon mRNA is only detected in hematopoietic tissues, including embryonic liver and adult bone marrow and spleen. Within the hematopoietic system, C/EBPepsilon is expressed primarily in myeloid cells, including promyelocytes, myelomonocytes, and their differentiated progeny. To identify potential functions of C/EBPepsilon, cell lines over-expressing the C/EBPepsilon protein were generated in the P388 lymphoblastic cell line. In contrast to the parental cell line, C/EBPepsilon-expressing cell lines displayed lipopolysaccharide-inducible expression of the interleukin-6 and monocyte chemoattractant protein 1 (MCP-1) genes as well as elevated basal expression of the MIP-1alpha and MIP-1beta chemokine genes. In the EML-C1 hematopoietic stem cell line, C/EBPepsilon mRNA levels increased as the cells progressed along the myeloid lineage, just preceding activation of the gene encoding the receptor for macrophage-colony-stimulating factor (M-CSFR). M-CSFR expression was stimulated in C/EBPepsilon-expressing P388 cell lines, when compared with either the parental P388 cells or P388 cell lines expressing either C/EBPalpha or C/EBPbeta. These results suggest that C/EBPepsilon may be an important regulator of differentiation of a subset of myeloid cell types and may also participate in the regulation of cytokine gene expression in mature cells.

Stem cell factor, the JAK-STAT pathway and signal transduction.

Recent work has demonstrated the importance of Janus family kinases (JAKs) and signal transducers and activators of transcription (STATs) in the stimulus-response coupling of receptors lacking intrinsic tyrosine kinase activity. In particular, the JAK-STAT pathway appears critical in signal transduction by interferon as well as numerous hematopoietic growth factors interacting with members of the hemapoietin receptor superfamily. Although ligands that interact with receptor tyrosine kinases (RTK), such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony stimulating factor-1 (CSF-1), have been shown to induce increases in phosphorylation of both JAKs and STATs, little is known about activation of this pathway by stem cell factor (SCF). This review will summarize what is known about the JAK/STAT pathway in relation to SCF signal transduction.

JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor.

Stem cell factor (SCF) is a hematopoietic growth factor that interacts with the receptor tyrosine kinase, c-kit. We have found that SCF-stimulates rapid and transient tyrosine phosphorylation of JAK2 in human and murine cell lines, as well as in normal human progenitor cells. JAK2 and c-kit were associated in unstimulated cells with further recruitment of JAK2 to the c-kit receptor complex after SCF stimulation. Treatment of cells with JAK2 antisense oligonucleotides resulted in a 46% decrease in SCF-induced proliferation. These data demonstrate that SCF induces tyrosine phosphorylation of JAK2 and suggest that JAK2 is a component of the SCF signal transduction pathway.

JAK2 is constitutively associated with c-Kit and is phosphorylated in response to stem cell factor.

Stem cell factor (SCF) interacts with the receptor tyrosine kinase c-Kit and has potent effects on hematopoiesis. We have examined the role of JAK2 in the SCF signal transduction pathway. JAK2 and c-Kit were constitutively associated, and treatment with SCF resulted in rapid and transient tyrosine phosphorylation of JAK2. Incubation of cells with JAK2 antisense oligonucleotides resulted in significant decreases in SCF-induced proliferation. These data suggest that JAK2 plays a role in SCF-induced proliferation.

Analysis of interleukin-2-dependent signal transduction through the Shc/Grb2 adapter pathway. Interleukin-2-dependent mitogenesis does not require Shc phosphorylation or receptor association.

The interleukin (IL)-2 receptor system has previously been shown to signal through the association and tyrosine phosphorylation of Shc. This study demonstrates that the IL-2 receptor beta (IL-2R beta) chain is the critical receptor component required to mediate this effect. The use of IL-2R beta chain deletion mutants transfected into a Ba/F3 murine cell model describes a requirement for the IL-2R beta "acid-rich" domain between amino acids 315 and 384 for Shc tyrosine phosphorylation and receptor association. COS cell co-transfection studies of IL-2R beta chain constructs containing point mutations of tyrosine to phenylalanine along with the tyrosine kinase Jak-1 and a hemagglutinin-tagged Shc revealed that the motif surrounding phosphorylated tyrosine 338 within the acid-rich domain of the IL-2R beta is a binding site for Shc. Deletion of this domain has previously been shown to abrogate the ability of IL-2 to activate Ras but does not affect IL-2-dependent mitogenesis in the presence of serum. Proliferation assays of Ba/F3 cells containing IL-2R beta chain deletion mutants in serum-free medium with or without insulin shows that deletion of the acid-rich domain does not affect IL-2-driven mitogenesis regardless of the culture conditions. This study thus defines the critical domain within the IL-2R beta chain required to mediate Shc binding and Shc tyrosine phosphorylation and further shows that Shc binding and phosphorylation are not required for IL-2-dependent mitogenesis. Neither serum nor insulin is required to supplement the loss of induction of the Shc adapter or Ras pathways, which therefore suggests a novel mechanism for mitogenic signal transduction mediated by this hematopoietin receptor.