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The human homologue of the Drosophila tumor suppressor lethal (2) tumorous imaginal discs (l(2)tid) gene, hTID1, encodes two proteins derived from alternate mRNA splicing. The splice variants TidL and TidS were previously reported from protein overexpression and dominant-negative mutant protein studies to exhibit opposing biological activities in response to exogenous cytotoxic stimuli. TidL was found to promote apoptosis while TidS suppressed it. To elucidate the physiological function of hTID1, we depleted hTID1 proteins using the technique of RNA interference (RNAi). Here, we show that cells essentially lacking expression of hTID1 proteins are protected from cell death in response to multiple stimuli, including cisplatin, tumor necrosis factor alpha/cycloheximide and mitomycin C. We also generated stable cell populations depleted of hTID1 proteins by RNAi using DNA vectors. In addition to apoptosis resistance, stable hTID1 knockdown cells exhibited an enhanced ability for anchorage-independent growth, as measured by an increase in soft-agar colony formation. These results suggest that hTID1 functions as an important cell death regulator and raise the interesting possibility that hTID1 could exert tumor suppressor activity.

Dysregulation of the centrosome duplication cycle has been implicated in tumorigenesis. Our previous work has shown that the human papillomavirus type 16 (HPV-16) E7 oncoprotein rapidly induces aberrant centrosome and centriole duplication in normal human cells. We report here that HPV E7-induced abnormal centriole duplication is specifically abrogated by a small molecule CDK inhibitor, indirubin-3'-oxime (IO), but not a kinase-inactive derivative. Importantly, normal centriole duplication was not markedly affected by IO, and the inhibitory effects were observed at concentrations that did not affect the G1/S transition of the cell division cycle. Depletion of CDK2 by siRNA similarly abrogated HPV E7-induced abnormal centrosome duplication and ectopic expression of CDK2 in combination with cyclin E or cyclin A could rescue the inhibitory effect of IO. IO treatment also reduced the steady-state level of aneuploid cells in HPV-16 E7-expressing cell populations. Our results suggest that cyclin/CDK2 activity is critically involved in abnormal centrosome duplication induced by HPV-16 E7 oncoprotein expression, but may be dispensable for normal centrosome duplication and cell cycle progression.

The WARTS gene encodes a kinase that localizes to the mitotic apparatus of a dividing cell. Named WARTS after the growths that develop in the eyes of Drosophila in which the gene is deleted. WARTS is also implicated as a tumor suppressor in mice and humans. In this issue of Oncogene, Iida et al. describe experiments suggesting that, in addition to a role in regulating mitosis, WARTS functions to prevent further rounds of DNA synthesis and mitosis in tetraploid cells. As well as opening up new possibilities of exploring the as yet ill-defined mechanistic basis of the tetraploidy checkpoint, the involvement of a tumor-suppressor gene in this checkpoint supports its importance as a safeguard against the acquisition of genomic instability, a key event in the progression to cancer.

ID1 is a member of the inhibitor of DNA binding/differentiation (ID) family of dominant negative helix-loop-helix transcription factors. ID-proteins have been implicated in the control of differentiation and transcriptional modulation of various cell cycle regulators and high levels of ID1 expression are frequently detected in various cancer types. However, it is unclear whether ID1 is a marker of highly proliferative cancer cells or whether it directly contributes to the tumor phenotype. A detailed analysis of ID1-expressing human cells revealed that a fraction of ID1 localizes to centrosomes. Ectopic expression of ID1 in primary cells and tumor cell lines resulted in accumulation of cells with abnormal centrosome numbers. There was no evidence for centrosomal localization or induction of centrosome abnormalities by the other ID family members. Hence, ID1 may contribute to oncogenesis not only by inhibiting transcriptional activity of basic helix-loop-helix transcription factors and abrogate differentiation but also by subverting centrosome duplication.

We previously described two human DnaJ proteins, hTid-1L and hTid-1S, which are derived from alternative splicing of the TID1 gene, the human homologue of the Drosophila tumor suppressor lethal(2) tumorous imaginal discs, and showed that hTid-1L promoted while hTid-1S antagonized apoptosis. There are two subsets of helper T cells, Th1 and Th2, of which Th2 cells are significantly less prone to apoptosis induced by stimulation through the T-cell receptor. This apoptotic process is known as activation-induced cell death (AICD). The molecular basis for the differential susceptibility of Th1 and Th2 cells to AICD is not known. Here we show that the antiapoptotic variant, Tid-1S, is selectively induced in murine Th2 cells following activation. Expression of a dominant-negative mutant of hTid-1S in a Th2 cell line strikingly enhanced activation of caspase 3 in response to CD3 stimulation, and caused the cells to become sensitive to AICD. Hence, the accumulation of Tid-1S in Th2 cells following activation represents a novel mechanism that may contribute to the induction of apoptosis resistance during the activation of Th2 cells.

The majority of human cancers are genomically unstable, often with gains or losses of whole chromosomes. In high-risk human papillomavirus (HPV)-associated cervical neoplasia, the two HPV-encoded oncoproteins E6 and E7 have been implicated in mitotic infidelity by their ability to induce centrosome-related mitotic disturbances. However, the mechanisms by which HPV E6 and E7 subvert centrosome homeostasis are strikingly different. Whereas the E7 oncoprotein rapidly drives centrosome duplication errors in cells that appear phenotypically normal, expression of the HPV E6 oncoprotein results in an accumulation of supernumerary centrosomes in multinucleated cells. The primary centrosome duplication defect in HPV E7 expressing cells may be linked to the ability of E7 to disrupt regulatory nodes that govern both the host cell division cycle machinery and the initiation of centrosome duplication. Most importantly, the E7 oncoprotein has been shown to dysregulate cdk2 activity, a major determinant for the initiation of centrosome duplication. HPV-induced centrosome abnormalities, multipolar mitoses, and aneuploidy often occur at early stages during cervical carcinogenesis and increase with malignant conversion. These findings suggest that HPV oncoprotein-induced chromosomal instability increases the risk for genetic changes that may ultimately facilitate carcinogenic progression.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPV-associated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular differentiation and proliferation in normal epithelium, thus allowing the virus to replicate in differentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely reflect this important function.

Tumor necrosis factor-alpha (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.