Identification of a novel splice variant of AML1b in ovarian cancer patients conferring loss of wild-type tumor suppressive functions.

Acute myeloid leukemia (AML) 1 is often disrupted by chromosomal translocations generating oncogenic fusions in human leukemias. However, its role in epithelial cancers has not been extensively investigated. Herein, we show a marked accumulation of AML1 transcripts including a high frequency of a novel alternatively spliced AML1b transcript lacking exon 6 (AML1b(Del179-242)) in ovarian cancer patients. The increases in RNA transcripts for total wild-type AML1 and AML1b(Del179-242) are associated with poor patient outcomes. We have shown that although both wild-type AML1b and AML1b(Del179-242) are localized to nuclear speckles, AML1b(Del179-242) was observed to have dramatically reduced transactivation potential with the plasminogen activator inhibitor-1 promoters and behaved as a weak dominant negative of wild-type AML1b. Wild-type AML1b was found to inhibit the growth of immortalized ovarian epithelial cells (T29) decreasing colony-forming ability. Moreover, we have identified a novel function of AML1b where it inhibits ovarian cell migration. In contrast, AML1b(Del179-242) has lost the ability to inhibit both ovarian cell proliferation and migration indicating that the functional effects observed with wild-type AML1b are dependent on amino acids 179-242. Collectively, these studies suggest that deregulated alternative splicing of AML1b transcripts may potentially contribute to the pathophysiology of ovarian cancers.

Genetic analysis of early- versus late-stage ovarian tumors.

In the United States, ovarian cancer is the fourth most common cause of cancer-related deaths among women. The most important prognostic factor for this cancer is tumor stage, or extent of disease at diagnosis. Although women with low-stage tumors have a relatively good prognosis, most women diagnosed with late-stage disease eventually succumb to their cancer. In an attempt to understand early events in ovarian carcinogenesis, and to explore steps in its progression, we have applied multiple molecular genetic techniques to the analysis of 21 early-stage (stage I/II) and 17 advanced-stage (stage III/IV) ovarian tumors. These techniques included expression profiling with cDNA microarrays containing approximately 18,000 expressed sequences, and comparative genomic hybridization to address the chromosomal locations of copy number gains as well as losses. Results from the analysis indicate that early-stage ovarian cancers exhibit profound alterations in gene expression, many of which are similar to those identified in late-stage tumors. However, differences observed at the genomic level suggest differences between the early- and late-stage tumors and provide support for a progression model for ovarian cancer development.

Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells.

Shc adaptor proteins play a role in linking activated cell surface receptors to the Ras signaling pathway in response to receptor mediated tyrosine kinase activation. While the function of Shc in the activation of the Ras pathway via binding to Grb2 has been well characterized, it is becoming increasingly apparent that Shc participates in additional signaling pathways through interactions with other cytoplasmic proteins. Using the yeast two-hybrid system, we have identified a unique Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes) with no similarity to other known proteins. mPAL binds specifically to the Shc SH2 domain and unlike previously described Shc SH2 domain-protein interactions, the association of mPAL and Shc is phosphotyrosine-independent. Both mPAL RNA and protein expression are restricted to tissues containing actively dividing cells and proliferating cells in culture. mPAL expression is induced upon growth factor stimulation and is down-regulated upon growth inhibition. This pattern, and timing of mPAL expression and its association with the Shc adaptor molecule suggests a role for this protein in signaling pathways governing cell cycle progression.

Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate.

BACKGROUND: Drosophila numb was originally described as a mutation affecting binary divisions in the sensory organ precursor (SOP) lineage. The numb gene was subsequently shown to encode an asymmetrically localized protein which is required for binary cell-fate decisions during peripheral nervous system development. Part of the Drosophila NUMB protein exhibits homology to the SHC phosphotyrosine-binding (PTB) domain, suggesting a potential link to tyrosine-kinase signal transduction. RESULTS: A widely expressed mammalian homologue of Drosophila numb (dnumb) has been cloned from rat and is referred to here as mammalian Numb (mNumb). The mNUMB protein has a similar overall structure to dNUMB and 67 sequence similarity. Misexpression of mNumb in Drosophila during sensory nervous system precursor cell division causes identical cell fate transformations to those produced by ectopic dNUMB expression. In vitro, the mNUMB PTB domain binds phosphotyrosine-containing proteins, and SH3 domains of SRC-family tyrosine kinases bind to mNUMB presumably through interactions with proline-rich regions in the carboxyl terminus. Overexpression of full-length mNUMB in the multipotential neural crest stem cell line MONC-1 dramatically biases its differentiation towards neurons, whereas overexpression of the mNUMB PTB domain biases its differentiation away from neuronal fates. CONCLUSIONS: Our results demonstrate that mNUMB is an evolutionarily conserved functional homologue of dNUMB, and establish a link to tyrosine-kinase-mediated signal transduction pathways. Furthermore, our results suggest that mNUMB and dNUMB are new members of a family of signaling adapter molecules that mediate conserved cell-fate decisions during development.

Cell cycle dependent regulation of the protein kinase TTK.

TTK is a novel protein kinase detectable in all proliferating human cells and tissues. Expression of the TTK gene is markedly reduced or absent in resting cells and in tissues with a low proliferative index. In view of the apparent association between TTK gene expression and cell proliferation, we examined the regulation of this protein kinase during transit of the cell cycle. We found very low levels of TTK mRNA and protein in starved cells. When cells are induced to enter the cell cycle, levels of TTK mRNA, protein and kinase activity increase at the G1/S phase of the cell cycle and peak in G2/M. TTK mRNA levels, as well as kinase activity, drop sharply in early G1, whereas protein levels are largely maintained. TTK may play a role in cell cycle control.

IL-2-induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression.

We have recently isolated the cDNA for a unique human 97-kDa kinase, TTK, by expression screening of a cDNA expression library using anti-phosphotyrosine antibodies. When expressed in Escherichia coli, TTK can phosphorylate serine, threonine, and tyrosine residues. Thus TTK appears to belong to a newly described family of kinases able to phosphorylate all three hydroxy amino acids. This family of multispecific kinases includes several other kinases involved in cell cycle progression. In support of a possible role in regulating cell cycle progression, TTK message is readily detected in rapidly proliferating tissues in vivo including testes, thymus, bone marrow, and many malignant tumors, but not in benign tissues with a low proliferative rate in vivo. To determine the effect of cell activation and cell cycle progression on TTK expression, we measured TTK mRNA and protein levels as well as kinase activity in freshly isolated T cells or IL-2-expanded T cell blasts activated to proliferate by the addition of a variety of mitogens. TTK mRNA levels, protein levels, and kinase activity were greatly enhanced when either freshly isolated PBL or T cell blasts were activated by cross-linking the TCR complex by mitogenic lectins or by bypassing the TCR with phorbol esters and cation ionophores. Incubation with IL-2 increased TTK expression in PBL blasts, which proliferate in response to IL-2, but not in fresh PBL, which do not proliferate in response to IL-2. TTK expression was blocked by either cyclosporin A or FK520, which inhibit IL-2 production and could be recovered by the addition of exogenous IL-2. Furthermore, TTK expression was prevented by incubation of the cells with rapamycin, which blocks IL-2 signaling. Thus, TTK expression in T cells appears to be a consequence of IL2-induced cell proliferation. Agonist-induced TTK expression was a delayed event occurring 12 to 24 h after activation of PBL blasts and 48 to 72 h after activation of fresh PBL. TTK protein and mRNA expression increased in both fresh PBL and T cell blasts concurrently with passage of cells through S phase as indicated by [3H]TdR incorporation and cell cycle analysis of propidium iodide-stained cells. TTK mRNA and protein levels reached a maximum as cells entered the G2 phase of the cell cycle. These results were confirmed by cell cycle blockade studies with aphidicolin and nocodazole wherein TTK protein levels are not detected in cells in G1 and are readily detectable in cells in the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic components of carcinogenesis.

Many of the genes encoding growth factors, growth factor receptors, enzymes, and other effector molecules that regulate normal cell growth are designated protooncogenes. Oncogenes, those genes associated with cellular transformation, differ from their protooncogenic progenitors by being mutated, overexpressed, or expressed at inappropriate times or locations in the cell. One of the activities of growth factors is to prime cells to undergo programmed cell death, which is characterized by a series of morphologic changes called apoptosis. In normal cells, specific mediators must be activated or suppressed to bypass programmed cell death. In tumor cells, either the pathways leading to apoptosis are not functional or the mediators that normally "rescue" cells from this fate are overexpressed or constitutively activated. In addition to the biochemical pathways that drive cell division, there are others that limit cell proliferation; these, designated tumor suppressors, anti-oncogenes, or recessive oncogenes, must be inactivated in normal cells to allow passage through the cell cycle and cell proliferation. In contrast to oncogenes, which are overexpressed or activated in tumors, tumor-suppressor genes are frequently inactivated in tumor cells, either by mutation or deletion. Thus, in normal cells a series of checks and balances must be overcome to allow initiation and continuation of cell division. In tumors, these processes are aberrant, resulting in increased rates of cell division, increases in the proportion of cells in the cell cycle, or increased survival of activated cells. Therefore, tumor cells frequently accumulate genomic alterations, which may result in the activation of a particular array of oncogenes, the inactivation of specific tumor-suppressor genes, and the bypassing of programmed cell death. Trials of antitumor agents that act by exploiting the overexpression of oncogenes in tumors and of the biochemical pathways by which they mediate cell proliferation are currently underway.

Transmembrane signaling by the interleukin-2 receptor: progress and conundrums.

Activation of the multicomponent interleukin-2 receptor (IL-2R) complex leads to a rapid increase in tyrosine phosphorylation of a number of cellular proteins including the IL-2R beta and IL-2R gamma chains of the IL-2R and the RAF-1 serine threonine kinase. In addition, phosphatidylinositol 3-kinase (PI-3K) protein and activity can be immunoprecipitated with anti-phosphotyrosine and anti-IL-2R beta antibodies from IL-2-activated but not resting T lymphocytes. We have demonstrated that the SH2 (SRC homology 2) domains of the 85 kDa subunit of PI-3K are sufficient to mediate binding of the PI-3K complex to tyrosine phosphorylated, but not non-phosphorylated IL-2R beta, suggesting that tyrosine phosphorylation is an integral component of the activation of PI-3K by the IL-2R. Since none of the members of the IL-2R complex contains an intrinsic tyrosine kinase domain, IL-2-induced tyrosine phosphorylation must be the consequence of activation of intracellular tyrosine kinases. SRC family members including lck, lyn and fyn have been demonstrated to associate with IL-2R beta through binding of the kinase domain to the acidic domain of IL-2R beta. However, we have demonstrated that the serine rich (SD) region of the cytosolic domain of IL-2R beta is also required for association of a tyrosine kinase with the IL-2R complex and that IL-2 can induce proliferation and tyrosine phosphorylation in cell lines which lack the known SRC family kinases expressed by T lymphocytes. Thus members of other kinase families besides SRC may also be involved in mediating IL-2 signal transduction. Biochemical studies and studies of cells expressing mutant IL-2 receptors indicate that IL-2-induced tyrosine kinase activation initiates a complex signaling cascade. The cascade includes SRC family kinase members such as lck, fyn, and lyn, activation of Raf-1 and PI-3K, and ras, and increased expression of the fos, fra-1, and jun protooncogenes. In addition, ligation of the IL-2R leads to rapid increases in myc expression and more delayed increases in the expression of the cdc2 and cdk2 kinases and the cyclins through a tyrosine phosphorylation independent pathway. Whether other biochemical processes initiated by IL-2R ligation, including activation of the MAP2, p70S6 and p90RSK serine threonine kinases, activation of NF-kappa B, and increased expression of Raf-1, Pim-1, bcl-2, IL-2R alpha and IL-2R beta, are consequences of the IL-2-induced tyrosine kinase cascade remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Neither the LCK nor the FYN kinases are obligatory for IL-2-mediated signal transduction in HTLV-I-infected human T cells.

IL-2 is one of the principal growth factors regulating the proliferation of T lymphocytes. Although two independent IL-2-binding molecules have been molecularly cloned and shown to participate in the formation of a high affinity receptor complex, their primary structures do not suggest a specific mechanism for IL-2 growth signal transduction across the cell membrane. Neither IL-2 receptor subunit contains an intrinsic kinase domain; nevertheless, tyrosine phosphorylation of various intracellular substrates is one of the first biochemical changes observed following activation of the IL-2 receptor (IL-2R). Both serine/threonine and tyrosine kinases can be co-precipitated as part of the IL-2R complex suggesting that the IL-2 signalling may involve the activation of non-covalently associated intracellular kinases. However, controversy exists as to which kinases are involved in IL-2 signal transduction; in particular, which kinase(s) mediates the first or proximal event(s) in the signalling process. Activation of the IL-2R leads to serine and threonine phosphorylation of the SRC tyrosine kinase family member, LCK, and an increase in LCK tyrosine kinase activity. Furthermore, LCK can be co-immunoprecipitated with the beta chain of the IL-2R indicating its association with the receptor complex. IL-2 has also been reported to increase FYN kinase activity and to alter its association with the 85 kDa subunit of phosphatidylinositol-3 kinase thus suggesting a role for FYN in IL-2 signal transduction. However, in this report, we now demonstrate that neither LCK nor FYN are obligatory for IL-2-induced growth of HTLV-I-infected human T cells. Lack of expression of LCK or FYN in the HTLV-I-infected T cell lines was demonstrated by a combination of Northern blotting, polymerase chain reaction, Western blotting, and in vitro kinase activity. Despite the absence of LCK or FYN, IL-2 induced similar patterns of rapid tyrosine phosphorylation. Similar results were observed in cell lines lacking expression of the LYN, FGR, HCK, and LTK tyrosine kinases. Thus, none of these tyrosine kinases alone appears to be required for growth signalling through the IL-2R in the HTLV-I-infected T cell lines analyzed. The findings raise the possibility that an, as yet, unidentified tyrosine kinase is involved. Alternatively, this biological signalling system may exhibit remarkable redundancy whereby several different tyrosine kinases may be capable of associating with the IL-2R complex and mediating intracellular signalling.

Expression of TTK, a novel human protein kinase, is associated with cell proliferation.

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.

T-lymphocyte proliferation: tyrosine kinases in interleukin 2 signal transduction.

Interleukin 2 (IL-2)-induced tyrosine phosphorylation appears to play a major role in IL-2-induced cellular proliferation. Several intracellular substrates including the beta chain of the IL-2 receptor complex (IL-2R beta), raf, MAP2 kinase, the regulatory 83 kDa subunit of phosphatidylinositol-3 kinase and S6 kinases are substrates for the IL-2 receptor activated kinase(s). However, none of the identified members of the IL-2 receptor complex exhibits intrinsic tyrosine kinase activity. Therefore, the IL-2R complex must activate intracellular tyrosine kinases. We have demonstrated that specific tyrosine and serine/threonine kinases are coprecipitated with IL-2 receptor constructs that mediate IL-2-induced cell proliferation but not with those that do not. The IL-2-activated tyrosine kinase appears to be associated with a serine and proline rich intracellular domain which is highly conserved between IL-2R beta and the erythropoietin receptor. Although the responsible kinase has not been identified, lck, fyn, fgr, ltk, hck and lyn can be ruled out as obligatory mediators. Using methods to clone tyrosine kinases from T cells, we have identified potential candidate kinases, including several which had not been known to be expressed by T lymphocytes as well as several unique kinases which had not been previously identified in any cell type.

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor.

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.