Indole-3-carbinol prevents PTEN loss in cervical cancer in vivo.

Indole-3-carbinol (I3C) is a phytochemical (derived from broccoli, cabbage, and other cruciferous vegetables) with proven anticancer efficacy including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. In a breast cancer cell line, I3C inhibited cell adhesion, spreading, and invasion associated with an upregulation of the tumor suppressor gene PTEN, suggesting that PTEN is important in inhibition of late stages in the development of cancer. The goal of this study was to determine the expression of PTEN during the development of cervical cancer and whether I3C affected expression of PTEN in vivo. We show diminished PTEN expression during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen. The implication is that loss of PTEN function is required for this transition. Additionally, dietary I3C increased PTEN expression in the cervical epithelium of the transgenic mouse, an observation that suggests PTEN upregulation by I3C is one mechanism by which I3C inhibits development of cervical cancer.

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells exposed to diindolylmethane in vitro.

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical cancer in animal model studies and in human clinical trials. I3C and its physiologic condensation product diindolylmethane (DIM) also induce apoptosis of tumor cells in vitro and in vivo, suggesting that these phytochemicals might be useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic acid microarray studies on transformed keratinocytes and tumor cell lines exposed to pharmacologic concentrations of DIM in vitro are consistent with a cellular response to nutritional deprivation or disruptions in protein homeostasis such as endoplasmic reticulum (ER) stress. In this report we investigate whether specific stress response pathways are activated in tumor cells exposed to DIM and whether the ER stress response might contribute to DIM's cytotoxicity. Induction of the stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78, GRP94, and asparagine synthase was documented by Western blot and real-time reverse transcriptase-polymerase chain reaction in C33A cervical cancer cells, and induction of a subset of these was also observed in cancer cell lines from breast (MCF-7) and prostate (DU145). The results are consistent with activation of more than 1 stress response pathway in C33A cells exposed to 75 microM DIM. Phosphorylation elF2alpha was rapidly and transiently increased, followed by elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional activation dependent on an ATF6-XBP-1 binding site was detected by transient expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT); induction of the GADD153 (CHOP) promoter was also confirmed by transient expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved at later times, coinciding with the onset of apoptosis. The results support the hypothesis that cytotoxic concentrations of DIM can activate cellular stress response pathways in vitro, including the ER stress response. Conversely, DIM was especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing degrees; nutrient limitation had a similar, but even more pronounced, effect. Because DIM toxicity in vitro is enhanced in cells undergoing nutritional deprivation and ER stress, it is possible that stressed cells in vivo, such as those within developing solid tumors, also have increased sensitivity to killing by DIM.

Requirement of STAT3 activation for differentiation of mucosal stratified squamous epithelium.

STAT3, a member of the signal transducers and activators of transcription (STAT) family, has been shown to play a key role in promoting proliferation, differentiation, or cell cycle progression, depending on cell type. A number of signaling pathways are altered in laryngeal papillomas, benign tumors induced by human papillomavirus 6/11. Papillomas overexpress the epidermal growth factor receptor and display enhanced MAP kinase and PI-3-kinase activity. They also show reduced activation of Akt and reduced levels of tyrosine-phosphorylated STAT3, due to overexpression of the tumor suppressor, PTEN. As papillomas show abnormalities in terminal differentiation, we examined the potential role of STAT3 in regulating epithelial differentiation. Laryngeal epithelial cells were suspended in supplemented serum-free medium. Differentiation was measured by Western blot analysis of keratin 13. Normal laryngeal epithelial cells were transfected with a constitutively active STAT3 or a dominant negative STAT3. Cells were transferred to suspension culture 24 h after transfection. Increased expression of keratin 13 was accompanied by the activation of STAT3 when differentiation was induced, and expression of a constitutively active STAT3 (STAT3C) enhanced the expression of keratin 13. In contrast, expression of a dominant negative STAT3 (Y705F) inhibited the expression of keratin 13. We conclude that activation of STAT3 is required for the differentiation of normal human stratified squamous epithelium.

BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation.

Fragmentation of polyploid megakaryocytes into platelets has great relevance for blood homeostasis. Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The antiapoptotic protein BclxL has been reported as up-regulated during megakaryocytic differentiation in vitro, but absent during late megakaryopoiesis. Our study focused on examining BclxL levels in megakaryocytes in vivo and in assessing the effect of its overexpression in transgenic mice (via the platelet factor 4 [PF4] promoter) on megakaryocyte development and platelet fragmentation. Interestingly, in the wild-type and less in PF4-driven transgenic mice, BclxL was not detected in a fraction of the large mature megakaryocytes, suggesting a regulation on the protein level. BclxL overexpression was associated with a moderate increase in megakaryocyte number, with no significant change in ploidy level or platelet counts. When the mice were challenged by induction of immune thrombocytopenia, the rate of platelet recovery was significantly slower in the transgenic mice as compared with controls. Moreover, proplatelet formation in vitro by transgenic megakaryocytes was limited. Transgenic megakaryocytes displayed poorly developed platelet demarcation membranes and cell margin extensions. Our study indicates that regulated expression of BclxL in megakaryocytes is important for the development of cells with a high potential to fragment into platelets.

PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells.

Laryngeal papillomas are caused by infection of the laryngeal epithelium by human papillomavirus type 6 or type 11 (HPV-6/-11). Previous studies in our laboratory have demonstrated an increase in PI3 kinase levels in papilloma tissue. However, activation of the downstream effector of PI3 kinase, protein kinase B (PKB/Akt), was reduced. This observation was explained by the elevated expression of the phosphatase and tensin homologue (PTEN), a recently characterized tumour suppressor, in papilloma tissue. Recent investigation of the possible functional roles of PTEN during papilloma development has now indicated that the level of tyrosine(705)-phosphorylated signal transducer and activator of transcription 3 [PTyr(705)STAT3] could be inversely correlated to that of PTEN as well. In vitro phosphatase assays suggested the presence of an increased level of a PTyr(705)STAT3 phosphatase in papilloma extract. Immunodepletion of PTEN from papilloma extracts resulted in a reduction of the PTyr(705)STAT3 phosphatase activity. Transfection of PTEN cDNA into HeLa cells attenuated STAT3 phosphorylation at Tyr(705) in a dose-dependent manner. This attenuation of STAT3 phosphorylation was independent of the STAT3 kinase. Interestingly, introduction of a lipid phosphatase mutant of PTEN (G129E) resulted in heightened PTyr(705)STAT3 phosphatase activity, relative to that obtained from wild-type PTEN transfection. These data indicate that PTEN negatively regulates STAT3 activation in HPV-infected papilloma cells. Induction of PTEN and reduction of activated STAT3 might be a result of a host defence mechanism or a virus-directed strategy to alter normal epithelial differentiation programming.

Signaling by the Mpl receptor involves IKK and NF-kappaB.

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.

Increased p50/p50 NF-kappaB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue.

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.