12/15-lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells.

The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells.

RB-dependent S-phase response to DNA damage.

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.

p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps.

The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun-N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38alpha(-/-) embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes.

The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells.

Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1.

Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells.

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.

Microinjection of anti-p21 antibodies induces senescent Hs68 human fibroblasts to synthesize DNA but not to divide.

Replicative senescence is characterized by irreversible growth arrest and has been defined by four genetic complementation groups. One of these groups is associated with the predominance of underphosphorylated, growth-suppressive retinoblastoma tumor suppressor protein (pRb). Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphorylate pRb, are underexpressed in senescent cells, others are expressed but inactive. This lack of cdk activity and arrest in the G1 phase of the cell cycle is likely attributable to the induction upon senescence of the G1-S cdk/cyclin inhibitors p21 (WAF1/CIP1/Sdi) and p16INK4. In fact, in early presenescent normal diploid fibroblasts in which p21 is inactivated, senescence is bypassed or postponed. Moreover, in senescent cells in which p53 function was inhibited, DNA synthesis was reinitiated, an effect likely attributable, in part, to the dependence of p21 expression on p53. We report here that the apparent inactivation of p21 in senescent human fibroblasts through the introduction of inhibitory alpha-p21 antibodies causes these cells to reenter the S-phase of the cell cycle. The disruption of p21 activity affects the p21-Rb-E2F pathway in that the expression of genes transcriptionally regulated by E2F, such as cyclin A and cdc2, were found to be up-regulated in injected cells. No evidence of cell division was observed. This suggests that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 activity is insufficient to permit cells to complete the cell cycle.

The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death.

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16ink4a, PSM-RB is also a potent inhibitor of cell cycle progression in RB-deficient tumor cells. Surprisingly, we identified a tumor cell line that is resistant to the cell cycle inhibitory effects of PSM-RB. This finding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this 'resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the first to show that RB inhibits cellular proliferation through at least two distinct mechanisms - inhibition of cell cycle progression and induction of cell death.

Retinoic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression.

Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor beta (RAR-beta) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-beta in the growth regulation of SCLC by retinoids, we restored RAR-beta expression in RAR-beta-negative H209 SCLC cells by retroviral transduction (H209-RAR-beta). We found that H209-RAR-beta, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-beta cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-beta cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-beta, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-beta cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-beta gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-beta.

Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.

Elevated cyclins and cyclin-dependent kinase activity in the rhabdomyosarcoma cell line RD.

An important early event in the differentiation of skeletal muscle cells is exit from the cell cycle, after which full expression of the muscle phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMS cells to differentiate or arrest, we studied the molecular response of the embryonal RMS cell line, RD, to culture in DM. Under these conditions, the retinoblastoma protein (RB) was primarily in the hyperphosphorylated state. This is in contrast to myoblasts cultured in DM, in which the hypophosphorylated form of RB is exclusively present. Measurements of the expression and activities of cyclin-dependent kinases (cdks) cdk2 and cdk4 indicated that RD cells maintained higher levels than do myoblasts, and the activity and abundance of these proteins did not significantly decrease upon culture in DM in RD cells, as they did in myoblasts. Similarly, elevated expression of cyclins D1, E, and A was observed in RD cells. Interestingly, cdk inhibitors are expressed in RD cells, with p16ink4 expression markedly elevated relative to myoblasts. Ectopic expression of p21cip1, p16ink4, or p27kip1 caused a growth arrest of RD cells but not detectable expression of a myogenic marker. Furthermore, a constitutively active RB protein could also inhibit the growth of RD cells without inducing myogenic differentiation. Taken together, these data suggest that the elevated levels of cdk2 and/or cdk4 observed in RD cells contribute to the inability of RD cells to growth arrest when cultured in DM but that these activities alone are not responsible for the failure of RD cells to differentiate.

Identification of ErbB3-stimulated genes using modified representational difference analysis.

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific contribution of each of the four family members to these processes remains unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to activation of ErbB3 and EGFR. The approach described is a modification of the representational difference analysis technique adapted for analysis of cDNA, which we have modified to permit identification of differential gene expression using as little as 20 microg of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplicons (small PCR-amplified fragments) which were subtracted against driver amplicons derived from unstimulated NIH-3T3 cells expressing the EGFR-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EGFR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons. Six of these, corresponding to known DNA sequences, were selected for further Northern blot analysis against total RNA prepared from the starting cell lines. Of these, the gene encoding the protein dlk (or a closely related protein, Pref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amyloid protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.

RalGDS functions in Ras- and cAMP-mediated growth stimulation.

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells.

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

Immunohistochemical evaluation of E-cadherin and epidermal growth factor receptor in non-small cell lung cancer.

Enhanced expression of the epidermal growth factor receptor and loss of expression of the cell-cell adhesion molecule E-cadherin have each been implicated in the development and progression of a variety of human malignancies. There is some evidence for a correlation between the expression of these two genes and the possible influence of the E-cadherin gene product on the expression of epidermal growth factor receptor. We evaluated 33 matched primary and metastatic non-small cell lung cancer specimens using immunohistochemical staining. There was a statistically significant correlation between staining intensity for epidermal growth factor receptor and E-cadherin in the primary tumors (P = 0.017, by Spearman correlation test). No difference was noted between primary and metastatic disease for either gene product. Studies that include clinical data are needed to clarify the significance of these findings and to evaluate whether these markers will help predict prognosis in tumors.

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

Enhanced E-cadherin expression in epidermal growth factor receptor expressing cells.

Expression of the epidermal growth factor receptor (EGFr) and the cell-cell adhesion molecule E-cadherin have individually been implicated in the biological activity of the most common human malignancies. There is also evidence for colocalization and for a correlation in the expression of these two proteins in human cells. To better define the relationship between these two gene products, we used immunohistochemistry and Western blot analysis to compare E-cadherin expression in various well characterized cell lines lacking expression of EGFr or expressing wild type, functional mutant or non functioning mutant EGFr. Parental NR6 cells, which lack endogenous EGFr, and a derivitive cell line NR6M721, which expresses EGFr lacking tyrosine kinase activity, showed low levels of E-cadherin expression with or without stimulation with EGF. In contrast, the derivative NR6c'973 cell line, which expresses an active EGFr defective in EGF induced internalization and down-regulation and NR6 cells expressing wild type EGFr showed strong E-cadherin expression. These results suggest that EGFr activation may regulate or enhance E-cadherin expression.

Immunolocalization of E-cadherin in human head and neck cancer.

Loss of expression of the cell-cell adhesion molecule E-cadherin has been shown to have a potential role in the dedifferentiation and progression of many human malignancies. We applied immunohistochemical staining for E-cadherin to eight formalin-fixed, paraffin-embedded primary and matched metastatic human head and neck carcinomas. In tumors that contained both well-differentiated and poorly differentiated components, staining was notably reduced in the poorly differentiated cells. Staining was nearly identical or only slightly reduced in metastases compared to primary tumors. As found in previous reports, E-cadherin expression may be involved in the dedifferentiation of these tumors.

c-Myc cooperates with activated Ras to induce the cdc2 promoter.

Expression of c-myc with constitutively active mutants of the ras gene results in the cooperative transformation of primary fibroblasts, although the precise mechanism by which these genes cooperate is unknown. Since c-Myc has been shown to function as a transcriptional activator, we have examined the ability of c-Myc and activated Ras (H-RasV-12) to cooperatively induce the promoter activity of cdc2, a gene which is critical for cell cycle progression. Microinjection of expression constructs encoding H-RasV-12 and c-Myc along with a cdc2 promoter-luciferase reporter plasmid into quiescent cells led to an increase in cdc2 promoter activity approximately 30 h after injection, a period which coincides with the S-to-G2/M transition in these cells. Expression of H-RasV-12 alone weakly activated the cdc2 promoter, while expression of c-Myc alone had no effect. Mutants of c-Myc lacking either the leucine zipper dimerization domain or the phosphoacceptor site Ser-62 could not cooperate with H-RasV-12 to induce the cdc2 promoter. These mutants also lacked the ability to cooperate with H-RasV-12 to stimulate DNA synthesis. Deletion analysis identified a distinct region of the cdc2 promoter which was required for c-Myc responsiveness. Taken together, these observations suggest a mechanistic link between the molecular activities of c-Myc and Ras and induction of the cell cycle regulator Cdc2.