Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.     

Evidence for Agrobacterium-induced apoptosis in maize cells

Agrobacterium spp. can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation. One major obstacle is that co-cultivation of Agrobacterium spp. with plant tissues often results in cell death. Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes. Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp. in maize tissues. p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas i.a.p. may act upstream to prevent their activation. This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells.     

Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.     

The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation.

Previous studies showed that the amino-terminal domain of Rous sarcoma virus p60v-src involved in myristylation and membrane association of the protein is required for morphological transformation and anchorage independence. Analysis of src delection mutants revealed that the amino-terminal one-third of p60v-src, including the membrane-binding domain, is not essential for induction of cell proliferation. These results demonstrated that, in contrast to the cellular target(s) involved in morphological transformation and anchorage independence, the target(s) involved in mitogenic activity is accessible to nonmyristylated src proteins.     

Specific desensitization of the epidermal growth factor receptor by pp60v-src

BALB/c 3T3 cells infected with a temperature-sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v-src tyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v-src exhibits a very rapid activation t1/2 (less than 5 min) of protein kinase activity on decreasing the temperature from 40 degrees C to 35 degrees C. This change in temperature was also found to induce a very rapid decrease in the affinity for 125I-EGF of receptors on the RSV-LA90-infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of 3H-bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v-src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactivation of pp60v-src.     

Suppression of ADAM17-mediated Lyn/Akt Pathways Induces Apoptosis of Human Leukemia U937 Cells: BUNGARUS MULTICINCTUS PROTEASE INHIBITOR-LIKE PROTEIN-1 UNCOVERS THE CYTOTOXIC MECHANISM*

Cell surface proteases have been demonstrated to play an important role in facilitating cell invasion into the extracellular matrix and may contribute significantly to extracellular matrix degradation by metastatic cancer cells. Abundant expression of these enzymes is associated with poor prognosis. Thus, protease inhibitors that repress cell surface proteases may be applicable to cancer therapy. Because soybean Kunitz-type trypsin inhibitor has been found to induce apoptotic death of human leukemia Jurkat cells, anti-leukemia activity of Bungarus multicinctus protease inhibitor-like protein-1 (PILP-1) is thus examined. PILP-1 induced apoptosis of human leukemia U937 cells, characteristic of loss of mitochondrial membrane potential, degradation of procaspase-8, and production of t-Bid. FADD down-regulation neither restored viability of PILP-1-treated cells nor attenuated production of active caspase-8 and t-Bid in PILP-1-treated cells, suggesting that the death receptor-mediated pathway was not involved in the cytotoxicity of PILP-1. It was found that PILP-1-evoked p38 MAPK activation and ERK inactivation led to PILP-1-induced cell death and down-regulation of ADAM17. Knockdown of ADAM17 by siRNA induced death of U937 cells and inactivation of Lyn and Akt. Immunoprecipitation suggested that ADAM17 and Lyn form complexes. Overexpression of ADAM17, LynY507F (gain of function), and constitutively active Akt suppressed the cytotoxic effects of PILP-1. PILP-1-elicited inactivation of Lyn and Akt was abrogated in cells with overexpressed ADAM17 or LynY507F. Taken together, our data indicate that ADAM17-mediated activation of Lyn/Akt maintains the viability of U937 cells and that suppression of the pathway is responsible for PILP-1-induced apoptosis.     

Inhibition of v-src-induced transformation by a GTPase-activating protein.

Previous work has shown that microinjection into cells of antibodies against p21ras blocks transformation by src, suggesting that oncogenic transformation by pp60v-src is dependent on p21ras. The activity of p21ras itself is regulated by its cyclic association with GDP-GTP, where p21ras-GTP is the active form and p21ras-GDP is the inactive form. A GTPase-activating protein (GAP) mediates the inactivation of p21ras by facilitating the conversion of the active p21ras-GTP to the inactive p21ras-GDP. This predicts that overexpression of GAP would inactivate p21ras and block transformation of cells by src. In this paper, we confirm this prediction. We report that overexpression of GAP in NIH 3T3 cells blocks transformation by pp60v-src but not by v-ras. Susceptibility to transformation by v-src is restored when GAP expression is lowered to levels comparable to that in control cells. These results support the suggestion that p21ras plays a central role in the signalling pathway used by pp60v-src.     

A perspective on mammalian caspases as positive and negative regulators of inflammation

Members of the caspase family of cysteine proteases coordinate the morphological and biochemical events that typify apoptosis. However, neutralization of caspase activity in mammals fails to block death in response to most proapoptotic stimuli. This is because many cell death triggers provoke mitochondrial dysfunction upstream of caspase activation as a consequence of BAX/BAK channel opening. Although genetic or pharmacological inactivation of caspases fails to block cell death in most instances, it does convert the phenotype from apoptosis to necrosis. This has important implications for how the immune system responds to such cells, as necrotic cells provoke inflammation whereas apoptotic cells typically do not. Here, we propose an alternative perspective on apoptosis-associated caspase function by suggesting that these proteases are activated, not to kill, but to extinguish the proinflammatory properties of dying cells. This perspective unifies the mammalian caspase family as either positive or negative regulators of inflammation.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.

The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

Protein kinase Cdelta regulates keratinocyte death and survival by regulating activity and subcellular localization of a p38delta-extracellular signal-regulated kinase 1/2 complex

Protein kinase Cdelta (PKCdelta) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKCdelta-dependent keratinocyte death. This study implicates p38delta mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKCdelta-dependent death signal. We show that coexpression of PKCdelta with p38delta produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G(1) cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKCdelta and p38delta and is not produced by replacing PKCdelta with PKCalpha or p38delta with p38alpha. A constitutively active form of MEK6, an upstream activator of p38delta, can also produce cell death when coupled with p38delta. In addition, concurrent p38delta activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38delta-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38delta-ERK1/2 complex relocates into the nucleus in response to PKCdelta expression. This regulation appears to be physiological, since H(2)O(2), a known inducer of keratinocyte apoptosis, promotes identical PKCdelta and p38delta-ERK1/2 activity changes, leading to similar morphological changes.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Conditional inactivation of Brca1 in the mouse ovarian surface epithelium results in an increase in preneoplastic changes

Epithelial ovarian cancer (EOC) is thought to arise from the ovarian surface epithelium (OSE); however, the molecular events underlying this transformation are poorly understood. Germline mutations in the BRCA1 tumor suppressor gene result in a significantly increased risk of developing EOC and a large proportion of sporadic EOCs display some sort of BRCA1 dysfunction. Using mice with conditional expression of Brca1, we inactivated Brca1 in the murine OSE and demonstrate that this inactivation results in the development of preneoplastic changes, such as hyperplasia, epithelial invaginations, and inclusion cysts, which arise earlier and are more numerous than in control ovaries. These changes resemble the premalignant lesions that have been reported in human prophylactic oophorectomy specimens from women with BRCA1 germline mutation. We also report that inactivation of Brca1 in primary cultures of murine OSE cells leads to a suppression of proliferation due to increased apoptosis that can be rescued by concomitant inactivation of p53. These observations, along with our finding that these cells display an increased sensitivity to the DNA-damaging agent cisplatin, indicate that loss of function of Brca1 in OSE cells impacts both cellular growth control and DNA-damage repair which results in altered cell behavior manifested as morphological changes in vivo that arise earlier and are more numerous than what can be attributed to ageing.     

Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: implication in the mechanism of apoptosis

Elevated levels of intracellular Ca(II) are a prominent feature of apoptosis, a natural form of cell death involved in many physiological and pathological processes. Serine proteases play crucial roles in apoptosis and have been implicated in the genomic DNA degradation and the massive protein degradation that occur during apoptosis. In this study, the effects of the elevated level of Ca(II) on the activity and inhibition of serine proteases were examined by spectrophotometric methods. The effects of the elevated levels of Ca(II), Mg(II), K(I), and Na(I) on the activity and inactivation of three representative members of serine proteases were determined. The level of serine protease activity in CEM-C7-14 leukemic cells was also evaluated in the presence and absence of dexamethasone-induced apoptosis, and also in the presence of A23187, a Ca(II)-ionophore. Among the four metal-ions studied, only Ca(II) was found to significantly enhance the activity of mammalian serine proteases. Ca(II) was also found to significantly protect the enzymes from inhibition, while the other three metal-ions showed no significant effect on the inactivation of the enzymes. Compared to the control sample, the enzymic activity was found to be higher during apoptosis, and in the presence of the Ca(II)-ionophore. Results of this study indicate that Ca(II) can significantly enhance the catalytic efficiency of serine proteases during apoptosis.