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Apoptosis and tumor cell death pathways in response to HAMLET

Hallgren, Oskar LU (2005)
Abstract (Swedish)
Popular Abstract in Swedish

Celldöd är en lika viktig process som celldelning. Under hela ens liv måste vävnadens storlek regleras och gamla eller skadade celler förnyas. Detta styrs av en process som kallas programmerad cell död. Denna process är noggrant reglerad så att enbart oönskade celler tas bort. Om regleringen sätts ur funktion leder detta till ohämmad celldelning och cancer. Ett problem med befintliga cancermediciner är att de inte är selektiva för tumörceller utan även dödar friska celler i den omgivande vävnaden. Vi har upptäckt en molekyl från bröstmjölk som selektivt dödar tumörceller men inte friska celler. Molekylen har vi döpt till HAMLET (Human alpha-lactalbumin made lethal to tumor cells). Molekylen är... (More)
Popular Abstract in Swedish

Celldöd är en lika viktig process som celldelning. Under hela ens liv måste vävnadens storlek regleras och gamla eller skadade celler förnyas. Detta styrs av en process som kallas programmerad cell död. Denna process är noggrant reglerad så att enbart oönskade celler tas bort. Om regleringen sätts ur funktion leder detta till ohämmad celldelning och cancer. Ett problem med befintliga cancermediciner är att de inte är selektiva för tumörceller utan även dödar friska celler i den omgivande vävnaden. Vi har upptäckt en molekyl från bröstmjölk som selektivt dödar tumörceller men inte friska celler. Molekylen har vi döpt till HAMLET (Human alpha-lactalbumin made lethal to tumor cells). Molekylen är ett komplex av ett protein, alfa-laktalbumin, och en fettsyra, oleinsyra. Alfa-laktalbumin är ett kalciumbindande protein som normalt har en kompakt struktur. Tar man däremot bort kalciumjonen öppnas strukturen upp. Vi tillverkar HAMLET just genom att ta bort kalciumjonen, men för att stabilisera och låsa fast proteinet i den öppna strukturen tillsätter vi fettsyran. Det är först när vi har bildat komplexet som molekylen kan inducera celldöd i tumörceller. Denna avhandling hade som syfte att försöka utreda hur HAMLET inducerar celldöd i tumörceller.



I studie I undersökte vi hur HAMLET inducerar celldöd i förhållande till den vanligaste formen av celldöd, apoptos. När en cell dör av apoptos så aktiveras en grupp av nedbrytande proteiner som kallas caspaser. Man har sett att många av de förändringar som sker vid apoptos kan härledas just till caspaser. HAMLET aktiverade flera olika caspaser men nivåerna var låga jämfört med andra stimuli. När vi blockerade aktiviteten hos caspaserna påverkade vi inte celldöden, vilket visar att HAMLET aktiverar andra celldödsvägar parallellt. En vanlig egenskap hos tumörceller är att kontrollproteiner som reglerar celldöd förlorar sin normala funktion genom mutationer. Detta är en av anledningarna till att tumörceller blir resistenta mot cancerbehandling. Vi undersökte därför hur två av dessa kontrollproteiner, p53 och Bcl-2, med förändrad funktion, påverkade celldöd inducerad av HAMLET. Vi såg ingen skillnad i känslighet mellan celler som hade dessa proteiner och kontrollceller, vilket visar att HAMLET kan kringgå de normala kontrollmekanismerna.



Studie II undersökte om HAMLET aktiverar en alternativ celldödsväg, autofagi, vilken inte är beroende av caspaser. HAMLET behandlade celler uppvisade alla de morfologiska förändringar som man förväntar sig vid denna form av celldöd. När vi blockerade denna form av celldöd dog cellerna i alla fall, vilket visar att HAMLET måste döda tumörceller via en annan celldödsväg.



Celldelning är också en mycket noggrant reglerad process. Celler går igenom olika cellcykelfaser där cellens innehåll dubbleras för att den sedan skall kunna dela sig. Ett särdrag hos tumörceller är att de kan dela sig hämningslöst. I studie III undersökte vi hur HAMLET påverkar celler i olika cellcykelfaser. När vi använde en dos som normalt dödar 50 % av cellerna dog celler i en viss fas, G1, mer än celler i de andra faserna, men då vi höjde dosen dödade HAMLET alla celler oberoende av cellcykelfas. De flesta celler i den mänskliga kroppen har en mycket specialiserad funktion vilket också kallas för att de är högt differentierade. Dessa celler delar sig inte speciellt ofta. Tumörceller däremot är ofta lågt differentierade och har en högre celldelningstakt. Vi undersökte därför hur känsliga tumörceller med olika differentieringsgrad var för HAMLET. Det visade sig att differentierade tumör celler var mindre känsliga för HAMLET. Denna studie visade att HAMLET till skillnad från andra celldödsstimuli dödar celler oberoende av cellcykelfas och att lågt differentierade tumör celler är känsligare för HAMLET än tumör celler som har en högre differentieringsgrad.



Studie IV undersökte om det är den strukturella förändringen i alfa-laktalbumin som är orsak till HAMLETs biologiska aktivitet. Alfa-laktalbumin i den öppna strukturen återgår normalt till den kompakta formen under de förhållanden som vi måste använda när vi behandlar tumörceller. Vi använde därför mutanter av alfa-laktalbumin som är fastlåsta i den öppna strukturen. Mutanterna hade ingen biologisk aktivitet på egen hand vilket visar att både proteinet och fettsyran behövs för den biologiska funktionen. (Less)
Abstract
HAMLET (Human alpha-lactalbumin made lethal to tumor cells) is a molecular complex of alpha-lactalbumin and oleic acid, that induces programmed cells death in tumor cells but leaves healthy differentiated cells unaffected. It has a broad anti-tumor activity as more than 40 cell lines from different origin are sensitive. The cell death activity of HAMLET is retained in vivo. HAMLET limits the progression of human glioblastomas in a rat xenograft model and reduces the lesion volume in skin papilloma patients. In HAMLET treated cells there are characteristic morphological changes of apoptosis. This thesis aimed to elucidate the cell death mechanism(s) activated by HAMLET.



A low apoptotic response was observed in HAMLET... (More)
HAMLET (Human alpha-lactalbumin made lethal to tumor cells) is a molecular complex of alpha-lactalbumin and oleic acid, that induces programmed cells death in tumor cells but leaves healthy differentiated cells unaffected. It has a broad anti-tumor activity as more than 40 cell lines from different origin are sensitive. The cell death activity of HAMLET is retained in vivo. HAMLET limits the progression of human glioblastomas in a rat xenograft model and reduces the lesion volume in skin papilloma patients. In HAMLET treated cells there are characteristic morphological changes of apoptosis. This thesis aimed to elucidate the cell death mechanism(s) activated by HAMLET.



A low apoptotic response was observed in HAMLET treated tumor cells, defined as caspase activity and phosphatidyl serine exposure. Caspase inhibition did not rescue cells from dying but it altered the mode of death. HAMLET triggered caspase dependent chromatin condensation but when caspases were blocked, alternative apoptosis like condensation was observed. Bcl-2 overexpression and p53 deletions or gain of function mutations did not influence the sensitivity to HAMLET. In conclusion, HAMLET induces cell death independently of caspases, Bcl-2 and p53.



In HAMLET treated tumor there is extensive vesicle formation typical for autophagy. Since autophagy has been described as a cell death mechanism we next investigated the relevance of autophagy in HAMLET induced cell death. Morphological changes characteristic of macro-autophagy was observed. There was as an increase of cytoplasmic vesicles enwrapped in double and multiple membranes. The vesicular content was shown to be acidic by MDC staining and the increase in acidic vesicle formation was reversed by 3MA, which prevents autophagy. LC3 was modified and translocated to autophagosomes in HAMLET treated cells and this response was reversed by 3MA. We identified damaged mitochondria that may be the trigger of this pathway. In parallel, there were indications of chaperon-mediated autophagy. An increase of HSC70 expression was observed and HAMLET colocalized with HSC70 in the cytoplasm. However, autophagy was not the cause of death as 3MA treatment did not rescue the HAMLET treated tumor cells from death. These results suggest that HAMLET triggers an autophagy in tumor cells, but this was not the mechanism of death.



As HAMLET kills tumor cells but not differentiated healthy cells we next investigated the influence of HAMLET on the cell cycle of tumor cells. In lymphoma cells, HAMLET preferentially killed G0/G1 at the LD50 concentration, but at higher doses all cells were killed regardless of cell cycle phase. The G2/M cells were the most sensitive to HAMLET in carcinoma cells, but at a high dose all cells were killed. Furthermore, the sensitivity to HAMLET decreased with the degree of differentiation. These results suggest that HAMLET differs from many cancer drugs by killing tumor cells regardless of cell cycle phase but with a preference for undifferentiated cells.



HAMLET consists of ?-lactalbumin in the apo state stabilized by the lipid cofactor oleic acid. The effect of the unfolded protein alone has not been tested. ?-lactalbumin rapidly reverts to the native state when exposed to physiologic solvent conditions, we therefore used ?-lactalbumin mutants that are retained in the apo state even at physiologic conditions. These proteins were not able to induce cell death, but could be converted to active complexes when they were combined with the fatty acid. These results show that both the protein and the fatty acid are required to form the active complex. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Wiman, Klas, Institutionen för Onkologi-Patologi, Karolinska Institutet, Stockholm
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biomedicinska vetenskaper (allmänt), General biomedical sciences, Klinisk biologi, apoptosis, autophagy, alpha-lactalbumin, Clinical biology, programmed cell death, HAMLET, cell cycle
pages
188 pages
publisher
Department of Laboratory Medicine, Lund University
defense location
Rune Grubb salen Sölvegatan 23 BMC
defense date
2005-12-21 09:00
ISSN
1652-8220
ISBN
91-85481-27-0
language
English
LU publication?
yes
id
c6721772-aa25-4a59-94eb-70829cc8f46d (old id 545990)
date added to LUP
2007-09-24 15:08:43
date last changed
2016-09-19 08:44:52
@phdthesis{c6721772-aa25-4a59-94eb-70829cc8f46d,
  abstract     = {HAMLET (Human alpha-lactalbumin made lethal to tumor cells) is a molecular complex of alpha-lactalbumin and oleic acid, that induces programmed cells death in tumor cells but leaves healthy differentiated cells unaffected. It has a broad anti-tumor activity as more than 40 cell lines from different origin are sensitive. The cell death activity of HAMLET is retained in vivo. HAMLET limits the progression of human glioblastomas in a rat xenograft model and reduces the lesion volume in skin papilloma patients. In HAMLET treated cells there are characteristic morphological changes of apoptosis. This thesis aimed to elucidate the cell death mechanism(s) activated by HAMLET.<br/><br>
<br/><br>
A low apoptotic response was observed in HAMLET treated tumor cells, defined as caspase activity and phosphatidyl serine exposure. Caspase inhibition did not rescue cells from dying but it altered the mode of death. HAMLET triggered caspase dependent chromatin condensation but when caspases were blocked, alternative apoptosis like condensation was observed. Bcl-2 overexpression and p53 deletions or gain of function mutations did not influence the sensitivity to HAMLET. In conclusion, HAMLET induces cell death independently of caspases, Bcl-2 and p53.<br/><br>
<br/><br>
In HAMLET treated tumor there is extensive vesicle formation typical for autophagy. Since autophagy has been described as a cell death mechanism we next investigated the relevance of autophagy in HAMLET induced cell death. Morphological changes characteristic of macro-autophagy was observed. There was as an increase of cytoplasmic vesicles enwrapped in double and multiple membranes. The vesicular content was shown to be acidic by MDC staining and the increase in acidic vesicle formation was reversed by 3MA, which prevents autophagy. LC3 was modified and translocated to autophagosomes in HAMLET treated cells and this response was reversed by 3MA. We identified damaged mitochondria that may be the trigger of this pathway. In parallel, there were indications of chaperon-mediated autophagy. An increase of HSC70 expression was observed and HAMLET colocalized with HSC70 in the cytoplasm. However, autophagy was not the cause of death as 3MA treatment did not rescue the HAMLET treated tumor cells from death. These results suggest that HAMLET triggers an autophagy in tumor cells, but this was not the mechanism of death.<br/><br>
<br/><br>
As HAMLET kills tumor cells but not differentiated healthy cells we next investigated the influence of HAMLET on the cell cycle of tumor cells. In lymphoma cells, HAMLET preferentially killed G0/G1 at the LD50 concentration, but at higher doses all cells were killed regardless of cell cycle phase. The G2/M cells were the most sensitive to HAMLET in carcinoma cells, but at a high dose all cells were killed. Furthermore, the sensitivity to HAMLET decreased with the degree of differentiation. These results suggest that HAMLET differs from many cancer drugs by killing tumor cells regardless of cell cycle phase but with a preference for undifferentiated cells.<br/><br>
<br/><br>
HAMLET consists of ?-lactalbumin in the apo state stabilized by the lipid cofactor oleic acid. The effect of the unfolded protein alone has not been tested. ?-lactalbumin rapidly reverts to the native state when exposed to physiologic solvent conditions, we therefore used ?-lactalbumin mutants that are retained in the apo state even at physiologic conditions. These proteins were not able to induce cell death, but could be converted to active complexes when they were combined with the fatty acid. These results show that both the protein and the fatty acid are required to form the active complex.},
  author       = {Hallgren, Oskar},
  isbn         = {91-85481-27-0},
  issn         = {1652-8220},
  keyword      = {Biomedicinska vetenskaper (allmänt),General biomedical sciences,Klinisk biologi,apoptosis,autophagy,alpha-lactalbumin,Clinical biology,programmed cell death,HAMLET,cell cycle},
  language     = {eng},
  pages        = {188},
  publisher    = {Department of Laboratory Medicine, Lund University},
  school       = {Lund University},
  title        = {Apoptosis and tumor cell death pathways in response to HAMLET},
  year         = {2005},
}