Divide et Impera mod for Total War: Rome IIDownload Found here: Moddb. General Info and Previews. Campaign Preview Threads. New Grand Campaign - - Character Traits Overhaul - - Buildings/Income Overhaul - - Politics. Battle Preview Threads. Battle Overhaul. Unit Preview Threads. Seleucids & Thrace - - Ptolemaic Kingdom - - Athens & Pontus - - Macedon & Epirus Part 1 - - Macedon & Epirus Part 2. Sparta - - Iweriu - - Kartli - - Colchis - - Bactria - - The Edetani - - Medewi - - Kaledonakoi. Atropatkan - - Greek Factions - - Lusitani - - Additional Units - - The Scordisci - - The Arevaci- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -Campaign Features. Population System- The general ideas of the system can be found in that thread and at the Po. R team's forums. All credit goes to their team for such a wonderful system. The basic concept is that population will be added to regions that will be used for recruitment. It is a bit more complicated than that, but you get the idea.- This will be the last major system we add to De. I. We already have reforms, supply and other smaller systems. With the addition of the population system, we are reaching a point where I can say the campaign is finally a somewhat deep and complex experience. That is something I never thought we could say when Rome 2 was launched New Grand Campaign Startpos and Start Date- Thanks to our partnership with the Ancient Empires team, we have had access to their work using the Augustus campaign map for the grand campaign as a foundation to work from for De. I. Because of this, we are happy to announce we will have a completely revamped Grand Campaign for our players to experience.- The campaign will begin in 2. BC rather than 2. BC. We have quite a few reasons for the move but, most importantly, we felt it would be something new for players who had been playing the previous grand campaign the past 3 years. The new start date allows us to incorporate the Pyrrhic Wars and creates a much more interesting starting situation in the central Mediterranean area.- We will also have the wonderfully designed emergent factions from the AE team. These factions are completely new entities that can be awoken or rebel in every single region on the map. This makes for a much more dynamic campaign experience as you can create friendly liberated tribes and kingdoms.- The Augustus map itself is an improvement as there are more regions, more factions and better designed provinces. Syracuse, Jerusalem and other cities are now walled capitals. There also seems to be better pathfinding on this map (like other scenario maps) compared to the original GC map.- Along with the new map, I will have the Assembly kit files available so that anyone can take them and make their own startpos and/or scenario mods for De. I. New Playable Factions, Unit Cards and Roster Overhauls- New unit cards are being designed by Rusty for use in all rosters. Total War ROME II Emperor Edition MULTi9-PROPHET 1- Unpack, burn or mount 2- Install the game 3- Copy the cracked content from PROPHET dir 4- Enjoy. Improves Rome: Total War in every aspect! It offers innovative AI enhancements, original gameplay improvements, new maps & campaigns, new units, new graphics. Total War: ROME II - Emperor Edition Create and Share Custom Mods Discover and download the best player-made mods for Total War: ROME II right here. Graphics and. They are amazing looking and you can already try some of them out in his submod thread!- Medewi (Meroe), Atropatkan (Media Atropatene) and Rhodes will be new playable factions. These also have received roster overhauls, with Rhodes focusing on naval warfare.- The Scordisci and Edetani will be playable and have received roster overhauls. These will replace Tylis and the Cantabri as playable factions, respectively.- Other roster overhauls include the Lusitani and the Arevaci. In addition, some factions will be receiving new units to differentiate them from other factions. Some of these units may replace existing units.- The Hellenic Overhaul is a massive endeavor that will redo many rosters including Sparta, Bactria, Athens, Pontus, Egypt, Epirus, Macedon and others. Income, Building Effects and New Buildings- The building effects in De. I have long been in need of some balance work. · M33t'n'fuck Year: 2009-2011, the Genre: Big breasts, Group sex, Anal sex, BDSM, Flash Censorship: None Developer: Meet And Fuck Games Platform: PC / Windows. Cornered Rat Software has announced that World War II Online is now available on Steam! On September 5, the game became available for all of North and South America. This guide tells you how to open a port on the Netgear AC1750 C6300. Punch, kick, hammer, hack, smash and shoot through hordes of enemies on a merciless mission for justice against the shadowy corporate forces you once worked for. We have many vanilla buildings but, also, a large amount of our own custom ones that have been added over the years. Now that we have had some time, we hope to go through and balance some of the effects to improve unused buildings and shake things up in terms of region management.- One major change will be the removal of a basic/hidden income bonus for all factions. This bonus exists in a large sum for every faction. Because of this, it is hard to truly impact income with in game actions. We will be moving most (if not all) of income to buildings. Now, raiding and sieging major economic centers will have a real impact on the enemy faction.- New special capital and port buildings will be added for historically important cities and ports. These will allow these cities to have special effects, garrisons and recruitment.- We are also working on a revamp of the building slot system so that we can have more freedom in the future to design region- specific and resource- specific buildings. Whether or not all of the new building ideas make it into 1.I am not sure. But, the idea will be that some resource regions will have special buildings.For example, a coin mint in a gold region or a timber camp in a timber region. Regional buildings will also allow us much more creative building chains in the future. Examples: Political System- We always wanted to make the political system a bit more interesting in Rome 2. Sadly, there seem to be some severe limitations in how much can be done here.- In 1. Rome and Carthage will still retain their 3 playables while the other factions will only have 1. However, with multiple parties, balancing political influence becomes more important and dynamic.- We hope to be able to mod and add to the existing dilemma and event system. Just how much can be done here is still a work in progress.- Its possible we will also look into making civil wars a more viable mechanic that can happen if political influence is not properly maintained. Historical This Year in History Messages- Thanks to the use of the R2. TR team's script, we will be having our own This Year in History messages. These have been researched and put together in order to add some flavor to the grand campaign as the turns go by.- Various historical events will be listed at the start of each year to let the player know what was occurring historically at the time and, perhaps, be able to compare it to how his or her campaign played out in a similar or different fashion. Trait System- We want some more RPG aspects to the game so we are playing with this idea. Rather than having 3 slots that may get used as you play, including random weird traits that seem to pop up for no reason, we want the in game actions to be meaningful. However, we also want to continue using our archetype system from 1. The idea here would be to have 3 traits at character creation. One is the character's political rank (all characters would start at rank 1). This would also feed into our political overhaul as it would immediately make each character a viable part of the system. The second trait is an "upbringing" or "educational" trait that would be random. This would reflect how that character grew up and the influences of early life on that character. Finally, the third trait would be our current personality archetype.- As the character makes certain in- game actions, that personality archetype could change based on those actions. So, eventually, a character who has a less- desirable archetype may become somewhat more virtuous through in- game actions. Perhaps, even, the reverse could happen as well.- Political ranks will no longer be purchased. Instead, those ranks must be earned through actions in the game, mostly based on battles won and age. Smaller Additions/Fixes- The CAI has been overhauled to make it much more aggressive and challenging.- Many other smaller fixes and additions will be included. Some you can see that are submods already like Flashheart's submod compilation. For example, raiding will impact siege holdout in the region, money from diplomacy will be reduced, culture conversion will be slowed, building conversion will be slowed, etc. As always, we look at ways to make the campaign a bit more realistic and .. Other small fixes that can already be found in the Po. R submod to the supply system and other reported bugs.- Reintroduction of client state levies for the player.- The mod pack itself has been completely reorganized so that tables are easily identified.
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Creeper was found an ARPANET. It was a worm that moved through modems to other systems where it displayed the message “I’M THE CREEPER : CATCH ME IF YOU CAN.” A similar program called Reaper followed Creeper. It appeared to attempt to find and delete Creeper. In 1. 97. 4 malware called Rabbit which multiplied so fast making copies of itself that systems crashed. In 1. 97. 5 a game written for the UNIVAC 1. Pervading Animal. The game asked questions in an attempt to determine what animal the user had thought of. The game, however, attempted to write itself to every writable program file, changing the creation time to be able to determine if it had already written to that file or not. It was never determined if this Trojan- like behavior was intentional or just an unintended bug. In the theoretical arena Jürgen Kraus wrote a master thesis called Selbstreproduktion bei Programmen (Self- reproduction of programs). Now, on to the modern history. The First Virus In The Wild. As described in Robert Slade’s history, the first virus in the wild actually predated the experimental work that defined current- day viruses. It was spread on Apple II floppy disks (which contained the operating system) and reputed to have spread from Texas A& M. Side note: Thanks to a pointer from anti- virus pioneer Fridrik Skulason we know the virus was named Elk Cloner and displayed a little rhyme on the screen: It will get on all your disks. It will infiltrate your chips. Yes it's Cloner! It will stick to you like glue. It will modify ram too. Send in the Cloner!For more info on Elk Cloner see the author’s (Richard Skrenta) page at: http: //www. The First Documented Experimental Virus Fred Cohen’s seminal paper Computer Viruses – Theory and Experiments from 1. From the paper…. On November 3, 1. The concept was first introduced in this seminar by the author, and the name ‘virus’ was thought of by Len Adleman. After 8 hours of expert work on a heavily loaded VAX 1. Unix, the first virus was completed and ready for demonstration. Within a week, permission was obtained to perform experiments, and 5 experiments were performed. On November 1. 0, the virus was demonstrated to the security seminar. Brain, PC- Write Trojan, & Virdem The common story is that two brothers from Pakistan (Basit Farooq Alvi and Amjad Farooq Alvi) analyzed the boot sector of a floppy disk and developed a method of infecting it with a virus dubbed “Brain” (the origin is generally accepted but not absolutely). Because it spread widely on the popular MS- DOS PC system this is typically called the first computer virus; even though it was predated by Cohen’s experiments and the Apple II virus. That same year the first PC- based Trojan was released in the form of the popular shareware program PC- Write. Some reports say Virdem was also found this year (it was presented to the underground Chaos Computer Club in Germany in December by Ralf Burger according to reports); it is often called the first file virus. File Infectors, Lehigh, & Christmas Worm The first file viruses started to appear. Most concentrated on COM files; COMMAND. COM in particular. The first of these to infect COMMAND. COM is typically reported to be the Lehigh virus. Cascade is reported to be the first self- encrypting virus. At this time other work was done to create the first EXE infector: Suriv- 0. Suriv = Virus backward). This virus evolved into the Jerusalem virus.) A fast- spreading (5. IBM mainframes during this year: the IBM Christmas Worm. And, in November, the Atari platform saw its first boot virus: SCA. Mac. Mag, Scores, & Internet Worm Mac.Mag, a Hypercard stack virus on the Macintosh is generally considered the first Macintosh virus and the Scores virus was the source of the first major Macintosh outbreak.Apple malware was not limited to the Macintosh however.This year, in June, saw the development of the Festering Hate Apple Pro.DOS virus. The Internet Worm (Robert Morris’ creation in November) causes the first Internet crisis and shut down many computers. CERT is created to respond to such attacks.AIDS Trojan This Trojan is famous for holding data hostage. The Trojan was sent out under the guise of an AIDS information program. When run it encrypted the user’s hard drive and demanded payment for the decryption key. Ghostballs is reported by some to be the first multipartite virus but it is a simple variation of the Vienna virus. VX BBS & Little Black Book (AT& T Attack) The first virus exchange (VX) BBS went online in Bulgaria. Here virus authors could trade code and exchange ideas. Also, in 1. 99. 0, Mark Ludwig’s book on virus writing (The Little Black Book of Computer Viruses) was published. While there is no proof, hackers are suspected of taking down the AT& T long- distance switching system. Based on the Vienna virus, the V2. P1 (alias 1. 26. 0) virus is sometimes considered to be the first polymorphic virus although the changes are minor. Tequila Tequila was the first serious polymorphic virus; it came out of Switzerland and changed itself in major ways in an attempt to avoid detection. Michelangelo, DAME, & VCL Michelangelo was the first media darling. A worldwide alert went out with claims of massive damage predicted on the March 6th trigger date. Actually, little happened. The same year the Dark Avenger Mutation Engine (DAME) became the first toolkit that could be used to turn any virus into a polymorphic virus. Also that year the Virus Creation Laboratory (VCL) became the first actual virus creation kit. It had pull- down menus and selectable payloads (though it’s reported to not have worked very well). Stealth_boot PMBSStealth_boot PMBS used a unique technique to operate. You caught it by booting from an infected floppy disk. Once installed, Stealth_Boot would install itself in extended memory, switched the computer into protected mode, and then ran a virtual V8. DOS and programs would use. Basically, the virus existed between the operating system and the hardware. Year of the Hacker, Concept. Hackers attacked Griffith Air Force Base, the Korean Atomic Research Institute, NASA, Goddard Space Flight Center, and the Jet Propulsion Laboratory. GE, IBM, Pipeline and other companies were all hit by the “Internet Liberation Front” on Thanksgiving. The first macro virus to attack Word, Concept, is developed. Boza, Laroux, & Staog. Boza is the first virus designed specifically for Windows 9. Laroux is the first Excel macro virus. 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When it is a PLATOON of Sinners, YOU HAVE NO IDEA the amount of DAMAGE that is being done. There are things that will seriously affect you, even after you have walked (or run) away. I would never have imagined that those on the industry suffer such ABUSE! I ASSUMED that the MEAL TICKET GOLDEN- GEESE were RESPECTED- based on my outdated research into CAL VISTA last Century (I read a few ADAM’S FILM WORLDs… just for the er, pictures!). The performers seemed to be well taken care of. Be that as it may, it appears that the current generation are not. I wonder how this could be, when some of the former performers have become producers? How could they stand by, and let that happen? The implications of this are disturbing. The Porn Industry makes astronomical amounts of money. The idea was that SATAN PAYS YOU VERY WELL, FOR THE LOSS OF YOUR SOUL. THAT was The DEAL! Everybody understood that!A girl, totally down on her luck, could always know, that if she were really THAT BAD, AND WORTHLESS, she could always do PORN- and make money, cunt over boob! She could always FEEL PROUD, that, as bad as things got, she NEVER sunk THAT low! However, it appears that even The Kingdom of Darkness is in Financial Recession now, and cannot afford to take care of Its victims. THAT MEANS that those in the Industry who are not Mega- Stars- and even those who are- are on a trap, which was always the case- but now, the fact is an 5. Gorilla in the room, which they cannot pretend not to see any more. I cannot believe that someone like Jenna Jameson will not be having recurring nightmares about losing EVERYTHING- not because she did something wrong, but because someone just turned legal age, who was more HER than HER- and she becomes redundant overnight!That every day she comes to work, she doesn’t step in the office, wondering if THAT is the day she is introduced to her REPLACEMENT??? I understand that there’s even a Drug Treatment for that! NOW WE GET TO THE REALLY FUN PART: EVERY person in the Porn Industry is part of a SODOM & GOMORRAH CITIZENRY, whereby ANGELS OF GOD are BUSY setting DEMOLITION CHARGES- and ranging their ARTILLERY and SNIPER NESTS- just waiting on the COMMAND- OF- GOD to just blow EVERY LAST MEMBER to Kingdom Gone.(“Heh, Heh, Heh! Well- that’s just TOO BAD! Good thing I never joined.”)EVERYBODY WHO HAS VIEWED PORNOGRAPHY IS PART OF THE PORNOGRAPHY INDUSTRY!(“WHAT???”)The Perceptual Data is a part of you now, FOR EVER. There are few documented wolf attacks on humans in North America in comparison to Eurasia and other larger carnivores. Search for Your Latest PC Games Here. Don’t Touch The Zombies Free Download PC Game; LEGO The Lord Of The Rings Free Download Full PC Game. No-registration upload of files up to 250MB. Not available in some countries. Tabtight professional, free when you need it, VPN service. Hey pls tell me when are you uploading railworks 3 :train simulator 2012 iam waiting from long time. Includes downloads, cheats, reviews, and articles. This domain name is for sale (100,000 USD): uploading.com Write us for more information @. This is a BLEMISH on your SOUL. It does not matter whether or not you viewed it for PERSONAL ENTERTAINMENT. It is IN you. WHAT are you DOING about it? Imagine that it were some other crime- BURGLARY, MUGGING- even JAYWALKING? Are you the type that walks on by, and DOES NOTHING- doesn’t “GET INVOLVED”? If you don’t warn a jaywalker, that what they are doing is wrong, maybe they will make someone swerve to avoid THEM, and hit someone you DO care about- YOU, perhaps? If your ENEMY’S OX is going astray, THE COMMANDMENT OF GOD says that IT IS YOUR RESPONSIBILITY to bring it back to him. EXODUS 2. 3: 4- 5. If thou meet thine enemy’s ox or his ass going astray, thou shalt surely bring it back to him again. If thou see the ass of him that hateth thee lying under his burden, and wouldest forbear to help him, thou shalt surely help with him.”IT IS AMAZING HOW PEOPLE WHO WANT TO JUDGE, CONDEMN, and EXECUTE those who break Commandments like. EXODUS 2. 0: 1. 3- 1. Thou shalt not Kill. Thou shalt not commit Adultery. Thou shalt not Steal. Thou shalt not Bear False Witness against thy Neighbour.”ARE MASSIVELY LAX AND MUTE ON THE TOPIC OF THE STRICT ENFORCEMENT OF THE 5. OTHER COMMANDMENTS, BY WHICH WE ARE JUDGED “SINNERS.”. JOHN 3: 4. 4. Whosoever committeth Sin transgresseth also so The Law: for Sin is the transgression of The Law. IN WESTERN CIVILIZATION ON GENERAL, AND IN AMERICA IN PARTICULAR, CHRISTIANITY is the most important component of Satan’s Kingdom. THEREFORE, you are NEVER going to defeat EVIL with CHTISTIANITY, because Satan has already PWNED every Church, since ACTS 1: 6. ACTS 1: 6. 6. When they were therefore cone together, they asked of him, saying,“Lord, wilt thou at this time RESTORE AGAIN THE KINGDOM TO ISRAEL?”WHAT WOULD I HAVE SAID, if I had been there? Obviously, something just as foolish- because I would be just as big an idiot as the people of the era. I have been blessed with 3. I did not deserve, to study this, and THEY only had 3 1/2. BUT- THE PERSON THEY WERE TALKING TO, has, with THEM as EYEWITNESSES, HEALED EVERY DISEASE they’d SEEN. He had Cast out Devils. He had RAISED THE DEAD. For his bother, HE HAD BEEN THOROUGHLY AND RELIABLY EXECUTED- pulled an “EDGE- OF- TOMORROW” RESURRECTION of his own, there. THEN HE HAD JUST SPENT 4. DAYS briefing them on The Secrets of The Universe- proving beyond all reasonable doubt that he was very much alive- despite UNBELIEVERS taking their best shot at killing him. THEN his own Disciples turn around, and say, to the effect, that: “If you don’t return POLITICAL AUTONOMY to the people who stood by and let you be MURDERED, then you haven’t done ANYTHING!”I FIND IT SIGNIFICANT, that JESUS DEPARTS FOR HEAVEN, immediately after this question is asked. The Christians of every Generation have always had a similar attitude. YOU HAVE IT. YOU HAVE ALWAYS THOUGHT that THE KINGDOM OF HEAVEN ON EARTH would only cone about if a guy in a white robe and a halo came down from the sky, blasted The Minons of Evil with X- MEN Cyclops Eyebeams. NO WAY you believed that IT IS ALL ON “YOU: ”IF you REALLY want THAT to happen- YOU DO IT. How can you END THE PORN INDUSTRY? By using ALL of your MIND. When you don’t worship God with ALL your MIND, how can you even FIND your whole mind? When you treat the PORNOGRAPHY Industry as something UNCONNECTED to YOU, and NOT- INSIDE- YOU, you cannot possibly DESTROY Pornography. The Teachers of Christianity are ASSETS/AGENTS of the OWNERS of The Porn Industry, and therefore GIVEN A CAREFULLY COMPOSED CURRICULUM to follow, that will GUIDE CHRISTIANS AWAY FROM THE ABILITY TO SOLVE THIS, AND SIMILAR PROBLEMS. People who boast of having ESCAPED from “THE MATRIX” are usually STILL IN THE MATRIX. Are you therefore ready to learn how to seriously destroy THE MATRIX, Sector- by- Sector? Download Bryan Adams - The Best Of Me (MP3@320) [FrankFoo] torrent from music category on Isohunt. Torrent hash: 717a886eee1cc101f7ebe8f91688bccb97e1662a. (lossy) » Скачать торрент (Pop) Bryan Adams - The Best Of Bryan Adams - 2004, MP3, 320 kbps Главная Трекер Поиск Правила. The Best Of Me 2. Here I Am. 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In human intestinal epithelial crypt (HIEC) cells, the PI3- K/Akt- 1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3- K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p. 85α, β, and p. C (p. 11. 0α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3- K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src- mediated suppression of anoikis.We report that: (1) the predominant PI3- K complexes expressed by HIEC cells are p.RNA- mediated expression silencing of p.Akt- 1 down- activation and consequent apoptosis; (3) the expression silencing of p.Akt- 1 down- activation and apoptosis; however, the impact of a loss of p.Akt- 1 activation and cell survival is significantly greater than that from the loss of p. Fak/Src signaling; however, the engagement of p. Src- dependent, whereas that of p. Fak- dependent (but Src- independent). Hence, HIEC cells selectively express PI3- K isoform complexes, translating into distinct roles in Akt- 1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src- mediated suppression of anoikis. Radioimmunotherapy of pancreatic cancer xenografts in nude mice using 90 Y-labeled anti-α 6 β 4 integrin antibody.The nonfibrillar type IV collagen is the major collagen found in basement membranes (BMs), forming network-like structures (Fig.Type IV collagen is made up of. Viruses successfully infect host cells by initially binding to the surfaces of the cells, followed by an intricate entry process.As multifunctional heterodimeric. Keywords: Anoikis, Fak, HIEC, PI3- K, Src, Survival. Keywords: Biomedicine, Virology, Cell Biology, Cancer Research, Oncology, Biochemistry, general. Introduction. Caspase- dependent apoptosis constitutes a complex and finely tuned process which performs crucial functions in development, tissue homeostasis and repair, as well as in the pathogenesis of several diseases [1–5]. It is now well understood that normal cells are intrinsically wired by default to undergo apoptosis and, consequently, require the input of signals in order to maintain apoptosis in a suppressed mode when not needed, or warranted [2–6]. Such critical cell survival signals are provided by various extracellular cues, including cell adhesion. To this effect, normal cells undergo caspase- dependent apoptosis by a process termed anoikis (a. In epithelial cells, those integrins belonging to the β1 subfamily are not only largely responsible for the establishment of a physical link between the extracellular matrix (ECM) and the cytoskeleton, but furthermore prevent the activation of the common anoikis pathway while at the same time driving the stimulation of various survival- promoting pathways [6–1. Hence, depending on the cell and tissue context, signaling originating from β1 integrins to promote cell survival and anoikis suppression will often implicate focal adhesion kinase (Fak; p. Fak), Src (p. 60. Src) and the phosphatidylinositol- 3 kinase (PI3- K)/Akt- 1 (PKB; p. Akt- 1) pathway [6–8, 1. The PI3- K/Akt signaling pathway is implicated in the regulation of various cell processes, including cell survival [1. The class I PI3- K consists of a complex that is formed by a catalytic (C) and regulatory (R) subunit [1. As a lipid kinase, PI3- K phosphorylates the substrate phosphatidylinositol (4,5) biphosphate (PIP2) to produce phosphatidylinositol (3,4,5) triphosphate (PIP3), which activates the effectors of the pathway (e. Akt) [1. 6–2. 0]. The engagement/activation of a PI3- K complex typically occurs through the recruitment/binding of an R subunit via its N- terminal SH2 domain, thus leading to a conformational change and consequent activation of its associated C subunit [1. With regards to integrin signaling, both Fak and Src have been shown competent in recruiting/engaging PI3- K in such a manner, whether directly or indirectly, depending on the cell type studied [6, 8, 1. Incidentally, three R (p. C (p. 11. 0α, p. 11. I PI3- K complexes [1. To this effect, there is increasing evidence that: (i) PI3- K isoform complexes can be selectively expressed and/or activated according to the cell type; and (ii) these isoform complexes can furthermore perform selective roles in the regulation of cell processes not only depending on the tissue context, but as well within the same given cell type [1. Much remains to be understood of the molecular determinants that regulate cell survival and death in intestinal epithelial cells (IECs), including their roles in the development of gastrointestinal disorders. In this respect, an improved comprehension of the specific signaling mechanisms that regulate survival and apoptosis/anoikis in normal human intestinal epithelial crypt (HIEC) cells is of direct relevance to the physiopathology of the gut, especially when considering that crypt alterations and epithelial apoptosis are regularly observed in inflammatory bowel disease, and that the persistence of aberrant crypt cells can lead to cancer [2. In previous studies, we have shown that the PI3- K/Akt- 1 pathway is not only critical for the survival of HIEC cells [2. Fak/Src- mediated signaling for anoikis suppression in a Fak- and Src- dependent manner [3. These observations are of interest, since a deregulation of Fak, Src and/or PI3- K/Akt survival signaling is often encountered in gastrointestinal cancers [1. Additionally, there is evidence that individual PI3- K R and/or C isoforms may be distinctively deregulated in gastrointestinal cancers [3. Hence, the still remaining open questions of the identity of the specific PI3- K isoform complexes that are responsible for driving HIEC cell survival, as well as those isoform complexes that are specifically engaged by integrin β1/Fak/Src- mediated signaling, have become quite germane. Consequently, in the present study, we investigated the expression of PI3- K isoforms and their roles in the integrin β1/Fak/Src- mediated regulation of cell survival and anoikis suppression in HIEC cells. We report herein that HIEC cells selectively express PI3- K isoform complexes, translating into distinct roles in cell survival, as well as in a selective engagement by Fak and/or Src within the context of integrin β1/Fak/Src- mediated survival signaling. Materials and methods. Materials. Specific antibodies directed against p. Fak, the phosphotyrosine. Fak (p. Y3. 97p. 12. Fak), the Src- phosphorylated tyrosine 5. Fak (p. Y5. 76/5. Fak), p. 57. Akt- 1, the phosphoserine. Akt- 1 (p. S4. 73p. Akt- 1), p. 60. Src, the phosphotyrosine. Src (p. Y4. 18p. 60. Src), and actin, were used as described previously [2. Abcam (Cambridge, CA), Cell Signaling Technology (Beverly, MA) and/or Millipore (Etobicoke, ON, Canada). Also used were specific antibodies directed to the following PI3- K C or R isoform subunits: p. Millipore; Cell Signaling Technology), p. Millipore), p. 11. Cell Signaling Technology), p. Millipore), p. 85α (Millipore), p. Abcam) and p. 55γ (Santa Cruz Biotechnology, Santa Cruz, CA). Note that the working functionality of each of the aforementioned PI3- K isoform antibody was verified and established by using protein lysates from granulocyte macrophage colony- stimulating factor (GMCSF)- stimulated human neutrophils (a kind gift from Patrick Mc. Donald, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada), which express all PI3- K isoforms studied herein [4.Other materials were purchased from Sigma (Oakville, ON, Canada) and/or Fischer Scientific (St- Laurent, QC, Canada), except where otherwise specified. Cell culture. The normal, non- transformed and non- immortalized HIEC- 6 cells, which exhibit all the morphological and functional properties of in vivo proliferative/undifferentiated human crypt enterocytes, have been extensively characterized elsewhere (as examples, see [2.HIEC- 6 cells were maintained and grown as already described [2. For experiments, cell cultures were maintained 2. M PIK- 7. 5 (Calbiochem, San Diego, CA), for the specific inhibition of p. PI3- K activity; (ii) 1. M TGX2. 21 (Calbiochem), for the specific inhibition of p. PI3- K activity; (iii) 1. M AS6. 05. 24. 0 (Tocris Bioscience, Ellisville, MO), for the specific inhibition of p. PI3- K activity; (iv) 1. M IC8. 71. 14 (Calbiochem), for the specific inhibition of p. PI3- K activity; (v) 3. M Ly. 29. 40. 02 (Calbiochem), for the “pan” inhibition of PI3- K activity enacted by p. M PF5. 73. 22. 8 (Tocris Bioscience), for the specific inhibition of Fak; (vii) 2. M PP2 (Calbiochem), for the inhibition of Src; (viii) 1. P4. C1. 0 (a kind gift of Erkki Ruoslahti, The Sanford- Burnham Medical Research Institute, La. Jolla, CA), which inhibits the binding activity of the β1 integrin subunit [2. |
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