jennifer james - omrf
TRANSCRIPT
puterbaugh foundation scholar
final research manuscripts
Jennifer James
Mentor: Hong Chen, Ph.D. Colleagues: Satish Pasula, Ph.D., Lucinda Maddera
Oklahoma Medical Research Foundation, Cardiovascular Biology Research Program
ABSTRACT The epsin group of proteins, conserved from yeast, is expressed in two main forms in mammals: epsin 1 and epsin 2. These proteins play key roles in
clathrin-mediated endocytosis and the activation of notch signaling. Specifically, the N-terminal of the protein containing ubiquitin interaction motifs (UIMs) allows epsin to regulate signal transmission of Vascular Endothelial Growth Factor (VEGF), a signal molecule that tumor cells use to stimulate angiogenesis. VEGFR-2 is the receptor protein for this signal molecule, and epsin is crucial in endocytosis that internalizes VEGFR-2, thus affecting the growth of tumors. Past breeding experiments have shown that mouse embryos with modi-fied genes which delete both forms of epsin are not viable past around E10, the early stages of organogenesis, due to disruption of notch signaling and vascular structure, but mice with only one of the two forms of epsin deleted experience no adverse phenotypic effects. Post-natal mice may be in-duced into “double knockouts” with drugs such as tamoxifen; this allows scientists to study the effects of a complete lack of epsin on the growth of various tumors. In this project we hypothesized that mice with a double knockout of epsin would experience inhibited melanoma and glioma tumor growth and angiogenesis, and we were particularly interested in the effects of an endothelial-cell-specific induc-ible knockout regulated by Vascular Endothelial Cadherin (VE-Cad). Both the VE-Cad knockout and the global tamoxifen knockout mice compared to wild type mice experienced inhibited tumor growth due to deregulated tumor angiogenesis, thus, leading to hypoxia of the tumor and apop-tosis of the tumor cells. These results reveal the role that epsin plays in the development and progression of tumors via controlling tumor angiogenesis.
INTRODUCTION Vascular Endothelial Growth Factor
(VEGF) is a signal molecule that stimulates blood vessel growth. Many malignant tumors use this molecule to stimulate angiogenesis and promote tumor growth and metastasis. Vascular Endothelial Growth Factor Receptor Two (VEGFR-2) is a tyrosine kinase receptor that often dimerizes with VEGF to initiate angiogenesis.1 Epsin is an endocytitic protein with two main mammalian iso-forms that regulates VEGF signaling by playing a role in forming the cur-vature of the clathrin-coated pits essential to clathrin-mediated endocy- tosis, a process that internalizes VEGFR-2.2- 3 Recent research has suggested, however, that epsin is not crucial to all clathrin-mediated endocy-tosis; the protein seems to function as a facilitator of endocytosis of ubiquiti- nated cargo, a role that is largely due to the ubiquitin interacting motifs (UIMs) located on the ENTH do-main on the N-terminal of the protein.3 The UIMs allow epsin to interact with ubiquitinated molecules such as those used in Notch signaling as well as receptor molecules such as VEGFR-2.4
Previous studies have showed that mouse embryos with a knockdown of both iso-forms of epsin were terminated in the early stages of organogenesis and demonstrated impeded Notch signaling and angio-genesis.3 Notch
signaling is a pathway that plays a part in cell division and apoptosis decision, and many tumors use the pathway to support angio- genesis.5 Through its role in the endocytosis of VEGFR-2, epsin serves as a regulator of VEGF sig-naling and angiogenesis in both pre-natal blood vessel formation and post- natal angiogenesis involved in both natural process such as the healing of wounds and malignant tumor growth. Although mice cannot fully develop with a double knockout of epsin isoforms, mice with only one of the two epsins are viable. When the gene cod-ing for the remaining epsin in these mice is sur- rounded by loxP sites, it can be disabled with ta-moxifen injections, yielding a post-natal mouse ex-pressing no forms of epsin.3 Such mice have no ad-verse phenotypic effects, and are useful in the study of the specific effect of epsin on malignant tumor growth. We hypothesized that both global knockouts deleting epsin from all cells and knockouts of Vascular Endothelial Cadherin (VE-Cad), deleting epsin only from endothelial cells, would inhibit tumor growth by deregulating an-giogenesis. To test this hypothesis, we implanted melanoma and glioma tu-mors in eighteen mice evenly divided into VE-Cad knockouts, global ta-moxifen knockouts, and wild type mice expressing both epsin proteins. The tumors were allowed to reach
a set volume, and then the mice were sacri- ficed. Tissue and tumor samples of the mice were analyzed using Western Blotting, as well as micros-copy staining techniques to analyze vasculature devel-opment, oxygenation of the tumors, and to verify the success of the knock- out. This data will be helpful in quantifying the effect of epsin on tumor vasculature as well as ex-ploring possible cancer treatments and clarifying the difference in tumor in- hibition between global tamoxifen and VE- Cad knockouts.
MATERIALS AND METHODS
Mice. Mice were raised in OMRF’s Live Animal Re-search Center (LARC). Mice were bred by 4h matings to be Cre positive and have the gene coding for epsin surrounded by loxP sites. Genotyping was conducted using qPCR to confirm the Cre positive. Knockout mice were injected with 10μL of tamoxifen (10mg/mL) dissolved in DMSO, and these injections continued every three days after the initial knockout. Mice were then given subcuta-neous injections of luciferase expressing IVC-B16/F10 Melanoma cells in DMEM. The mice were anesthetized with isofluorane for each injec- tion contained 1x106 cancer cells. The mice were then monitored for tumor growth, and once developed the tumors were measured
every two to three days. Tumor vol-ume was extrapolated using the formula length times width squared time 0.532. When any diameter of the tumor exceeded 1.5 cm in length, the mouse was sacrificed.
Western Blotting. Western Blot analysis was conducted on brain, heart, kidney, liver, lung, and spleen tissue harvested from the sacrificed mice. Tissue was homogenized in 87%lysis buffer with 10% triton, 2% 0.5M NaVO4, 1% 100XNEM, and 1 protease inhibitor tablet. These tissue sam-ples were then run on a 7.5% SDS PAGE gel and transferred to nitrocellu-lose membrane, which was cut into strips according to the range marker. The strips
were blocked in 5% milk in Tris Buffered Saline with Tween-20 (TBST) for 45 minutes. The strips were then incubated in
primary antibodies rabbit α epsin diluted 1:4000 in TBST and mouse α GapdH di- luted 1:5000 in TBST overnight at 4ºC. The strips were washed in TBST and incubated in goat α rabbit IgG and goat α mouse IgG both diluted 1:5000 in TBST for one hour at room temperature. The blots were then developed.
Hypoxia Staining. Sections of tumor from the sacrificed mice were imbedded in paraffin for later microscopy. These slides were later deparaf-finized, and antigen retrieval was conducted in 0.05% Pronase in PBS at 40ºC. Primary antibody for this stain was Hy-poxyprobe-1MAb1 diluted 1:50 in PBS, and secondary was Biotin- conjugated F (ab’)2 rabbit α mouse IgG diluted 1:500 in Phosphate Buffered Saline (PBS). DAB kit was added, and the slide was
counter-stained with Hematoxylin. These slides were mounted with Permafluor and viewed on a light micro-scope with a 4X objective. Quantitative analysis of hypoxia was conducted by comparing color threshold masks of tumor samples with and without primary antibody. The masks were created in ImageJ, and ana-lyzed to yield the percent of the sample area that displayed the stain color associated with hypoxia. The difference between the unstained and the stained sections repre-sented the total percentage of cells suffering hypoxia in the sample.
TUNEL Staining. Sections of tumor from the sacrificed mice were imbedded in paraffin for later microscopy. These slides were later deparaf-finized, and antigen retrieval was conducted in Citric Acid Solution with Tween-20 brought to a boil. Slides were
Figure 2. Western Blot Analysis shows the relative protein amounts of epsin and GapdH in VE-Cad knockout tissue. GapdH serves as a control with equal protein amounts, and epsin 1 is noticeably knocked-down in KO tissue, particularly liver and spleen tissues.
Figure 1. Wild Type Mice in both injection batches developed larger tumors more quickly than VE-Cad or Tamoxifen knockout mice.
incubated in TUNEL solution con-sisting of 45 μL Label Solution and 5μL enzyme for one hour in darkness, and slides were mounted in Permafluor and viewed under a confocal micro-scope using a 10X and 20X objective. Quantitative analysis was conducted as described for the Hypoxia slides.
CD-31 Staining. Mice were injected with FITC-Dextran 30 minutes prior to sacrific- ing. Tumor samples were harvested and preserved in O.C.T embedding medium and flash frozen in liquid nitrogen. Samples were cut 30 microns thick and fixed on slides. They were then blocked in PBS with 3% Bovine Serum Albumin (BSA), 3% donkey serum and 0.3% triton. Primary antibody was CD-31 di-luted 1:4 in PBS with triton and 1% BSA. Secondary antibody was donkey α rat AF594 in PBS. Slides were fixed in 4% paraformalde-hyde and mounted with Permafluor and viewed under a confocal
fluores-cent microscope using the 20X and 40X oil immer-sion objectives.
RESULTS The tumors in the knockout mice tended
to develop much more quickly than those in the wild type mice. Figure 1 shows the growth of total tumor volume of global tamoxifen, VE-Cad and wild type mice. The mice were injected in two batches approximately two weeks apart. After almost two months, the global tamoxifen knockout mice of the first batch have yet to develop even one tu-mor. All of the other mice in this group have been sacrificed with large tu-mors. Although the VE-Cad mice did develop tu-mors significantly before the tamoxifen knockout mice, they also showed signs of tumors well after the wild type mice. This confirms our hypothesis that knockout mice would experience inhibited tumor growth,
and the rest of the experiment was designed to provide insight into what mechanisms caused this inhibition.
Western Blotting of the VE-Cad knockout mice confirmed that the knock-out of epsin is not uniform throughout body tissue. In our mice, the ep-sin knockout was most obvious in the brain and liver (Fig. 2). This confirms the physiological difference between the VE- Cad and Global Tamoxifen knock-out mice and provides a likely explanation for the difference between global and endothelial cell spe-cific knockout tumor growth.
The TUNEL and Hypoxia staining procedures highlight cell death and lack of oxygen, respec-tively. Our Hypoxia stain- ing demonstrated a significant increase in cells lacking oxygen in knockout mice. The TUNEL stain-ing, similarly, highlighted large areas of dead cells along the outer edges of knockout tumors, while wild type tumors exhibited only limited and scattered cell death. Finally, CD-31 staining confirmed the de-regulation of angiogenesis. The knockout tumor sam-ples stained with CD- 31 exhibited much thicker and disorganized vasculature, while the vessels in the wild type samples tended to be organized and evenly spaced. This confirms our hypothesis that epsin is needed to keep vasculature growth regulated.
DISCUSSION
Our data confirmed that epsin knockouts inhibit malignant melanoma and glioma tumor growth. When monitoring the mice, we discovered that the wild type mice devel-oped tumors much more quickly than the knockout mice (Fig.1). The mice were generally sacrificed when the longest tumor diameter exceeded 1.5cm, but in some cases a knock-out tumor sample was needed to compare to wild type tumor data, and a knockout mouse had to be sacrificed before reaching the maximum allowed tu- mor size. This influenced our final tumor volume measures, but the slowed growth pattern of tumors in mice not expressing ep- sin remained obvious. The tumor growth in mice with a global tamoxifen knock-out of epsin was slower than the VE-Cad knockout tumor growth as well as the wild type tumor growth. This is likely re-lated to the Western Blotting results, which confirmed that VE- Cad mice did not experience a total knockout of epsin in all major organs. Tumor growth seems to correlate directly with the extent of the epsin knockout in these experiments.
Interestingly, none of the global tamoxifen knockout mice from the first batch of mice
Figure 3. Tumor vasculature in knockout (KO) mice is denser, but less organized than that of wild type (WT) mice as depicted by CD-31 fluorescent staining.
Figure 4. TUNEL staining causes dead cells to glow green under a fluorescent microscope. Tumors in knockout mice show greater rates of cell death than those in wild type mice.
receiv-ing tumors developed a tumor in the first two months of monitoring. The cells implanted in these mice were from the same culture as the cells implanted in the VE-Cad and wild type mice, all of which developed tumors large enough to sacrifice the mouse. One of these mice was infected with glioma tumor cells, and the other two were in-jected with melanoma cells. In the second batch of mice, those injected with tumor cells on June 9, one of the tamoxifen knockout mice developed a tumor in the first 40 days compared to two VE-Cad knockout mice and three wild type mice. We were surprised by the extent of the tumor inhi-bition in the knockout mice, but the result is strong evidence of the role epsin plays in tumor angiogenesis.
Although the death by suffocation of tumors with very dense vascula-ture may seem counterintuitive, the re-sults of our experiment reveal that without epsin angiogenesis can, in fact, perpetuate to a point that the new vessels choke, rather than oxygenate the tumor. CD-31 staining re-veals that the knockout mice actually developed more, and sometimes larger, blood vessels, but the FITC-Dextran in-jected into the mice was unable to penetrate these new vessels (Fig. 3). Blood is likewise unable to reach the tumor through these vessels; they are useless to the tu-mor.
Staining procedures revealed that cell death, particularly along the edges of the tumors, in-creased dramatically in knockout mice (Fig. 4). These cells at the edges of the tumors also showed signs of hypoxia, or lack
of oxygenation. These re-sults are indicative of the disruptive vasculature caused by the deregulation of angiogenesis. The dis-ordered growth of blood vessels to the tumor causes a lack of oxygen, particularly around the edges of the tumor, where the cells eventually die. This reduces the possibil-ity of tumor growth and metastasis.
Overall, the results of our experiment suggest that the amount of epsin in an organism has a defi-nite impact on the growth of tumor cells in that or-ganism. Any amount of epsin deletion can signifi- cantly slow tumor progression, and complete deletions of epsin provide striking results. The corre-lation between epsin, Notch signaling, and an-giogenesis is an important one in understanding the progression of one of the world’s most famous killers.
ACKNOWLEDGMENTS This project could not have been completed
without the support of the Oklahoma Medical Re-search Foundation, and funding from the Puter-baugh Foundation. Dr. Hong Chen has been an incredible resource, and I
am extremely grateful to her for the chance to par-ticipate in her research this summer. The mem-bers of Dr. Chen’s lab have been helpful and pleasant to work with. I would especially like to thank Dr. Satish Pasula, Cindy Maddera, and John McManus for taking the time to walk me through experiments as well as Laurencia Trujillo and Christine Trahms for sharing this experience with me.
REFERENCES 1. Anna-Karin Olsson, Anna Dimberg, Jo-
han Kreuger and Lena Claesson-Welsh 2006 VEGF receptor sig-naling−in control of vascular function. Na-ture Reviews / Mole-cular Cell Biology, VOL. 7, PG. 359.
2. Hong Chen, Silvia Fre. Valdimir I. Slep-nev, Maria R. Capua, Kohji Takei, Margaret H. Butler, Pier Paolo Di Fiore, Pietro De Camilli 1998 Epsin is an EH-domain-binding protein impli- cated in clathrin mediated endocytosis. Nature, vol. 394, pg. 793.
3. Hong Chen, Gevevieve Ko, Ales-san- dra Zatti, Giuseppina Di Gia-como, Lijaun Liu, Elisabetta Raiteri, Ezio Perucco, Chiara Collesi, Wang Min, Caroline Zeiss, Pietro De Camilli, Ot- tavio Cremona. Embryonic arrest at midgestation and disruption of Notch signaling pro-duced by the absence of both epsin 1 and epsin 2 in mice. Proc Natl Acad Sci U S A., 2009 (In Press).
4. Hong Chen and Pie-tro De Camilli. The association of epsin with ubiquit- inated cargo along the endo-cytic path- way is negatively regulated by its inter- action with clathrin. Proc Natl Acad sci U S A, 2004 (In Press).
final research manuscripts
Jennifer James
Mentor: Hong Chen, Ph.D. Colleagues: Satish Pasula, Ph.D., Lucinda Maddera
Oklahoma Medical Research Foundation, Cardiovascular Biology Research Program
ABSTRACT The epsin group of proteins, conserved from yeast, is expressed in two main forms in mammals: epsin 1 and epsin 2. These proteins play key roles in
clathrin-mediated endocytosis and the activation of notch signaling. Specifically, the N-terminal of the protein containing ubiquitin interaction motifs (UIMs) allows epsin to regulate signal transmission of Vascular Endothelial Growth Factor (VEGF), a signal molecule that tumor cells use to stimulate angiogenesis. VEGFR-2 is the receptor protein for this signal molecule, and epsin is crucial in endocytosis that internalizes VEGFR-2, thus affecting the growth of tumors. Past breeding experiments have shown that mouse embryos with modi-fied genes which delete both forms of epsin are not viable past around E10, the early stages of organogenesis, due to disruption of notch signaling and vascular structure, but mice with only one of the two forms of epsin deleted experience no adverse phenotypic effects. Post-natal mice may be in-duced into “double knockouts” with drugs such as tamoxifen; this allows scientists to study the effects of a complete lack of epsin on the growth of various tumors. In this project we hypothesized that mice with a double knockout of epsin would experience inhibited melanoma and glioma tumor growth and angiogenesis, and we were particularly interested in the effects of an endothelial-cell-specific induc-ible knockout regulated by Vascular Endothelial Cadherin (VE-Cad). Both the VE-Cad knockout and the global tamoxifen knockout mice compared to wild type mice experienced inhibited tumor growth due to deregulated tumor angiogenesis, thus, leading to hypoxia of the tumor and apop-tosis of the tumor cells. These results reveal the role that epsin plays in the development and progression of tumors via controlling tumor angiogenesis.
INTRODUCTION Vascular Endothelial Growth Factor
(VEGF) is a signal molecule that stimulates blood vessel growth. Many malignant tumors use this molecule to stimulate angiogenesis and promote tumor growth and metastasis. Vascular Endothelial Growth Factor Receptor Two (VEGFR-2) is a tyrosine kinase receptor that often dimerizes with VEGF to initiate angiogenesis.1 Epsin is an endocytitic protein with two main mammalian iso-forms that regulates VEGF signaling by playing a role in forming the cur-vature of the clathrin-coated pits essential to clathrin-mediated endocy- tosis, a process that internalizes VEGFR-2.2- 3 Recent research has suggested, however, that epsin is not crucial to all clathrin-mediated endocy-tosis; the protein seems to function as a facilitator of endocytosis of ubiquiti- nated cargo, a role that is largely due to the ubiquitin interacting motifs (UIMs) located on the ENTH do-main on the N-terminal of the protein.3 The UIMs allow epsin to interact with ubiquitinated molecules such as those used in Notch signaling as well as receptor molecules such as VEGFR-2.4
Previous studies have showed that mouse embryos with a knockdown of both iso-forms of epsin were terminated in the early stages of organogenesis and demonstrated impeded Notch signaling and angio-genesis.3 Notch
signaling is a pathway that plays a part in cell division and apoptosis decision, and many tumors use the pathway to support angio- genesis.5 Through its role in the endocytosis of VEGFR-2, epsin serves as a regulator of VEGF sig-naling and angiogenesis in both pre-natal blood vessel formation and post- natal angiogenesis involved in both natural process such as the healing of wounds and malignant tumor growth. Although mice cannot fully develop with a double knockout of epsin isoforms, mice with only one of the two epsins are viable. When the gene cod-ing for the remaining epsin in these mice is sur- rounded by loxP sites, it can be disabled with ta-moxifen injections, yielding a post-natal mouse ex-pressing no forms of epsin.3 Such mice have no ad-verse phenotypic effects, and are useful in the study of the specific effect of epsin on malignant tumor growth. We hypothesized that both global knockouts deleting epsin from all cells and knockouts of Vascular Endothelial Cadherin (VE-Cad), deleting epsin only from endothelial cells, would inhibit tumor growth by deregulating an-giogenesis. To test this hypothesis, we implanted melanoma and glioma tu-mors in eighteen mice evenly divided into VE-Cad knockouts, global ta-moxifen knockouts, and wild type mice expressing both epsin proteins. The tumors were allowed to reach
a set volume, and then the mice were sacri- ficed. Tissue and tumor samples of the mice were analyzed using Western Blotting, as well as micros-copy staining techniques to analyze vasculature devel-opment, oxygenation of the tumors, and to verify the success of the knock- out. This data will be helpful in quantifying the effect of epsin on tumor vasculature as well as ex-ploring possible cancer treatments and clarifying the difference in tumor in- hibition between global tamoxifen and VE- Cad knockouts.
MATERIALS AND METHODS
Mice. Mice were raised in OMRF’s Live Animal Re-search Center (LARC). Mice were bred by 4h matings to be Cre positive and have the gene coding for epsin surrounded by loxP sites. Genotyping was conducted using qPCR to confirm the Cre positive. Knockout mice were injected with 10μL of tamoxifen (10mg/mL) dissolved in DMSO, and these injections continued every three days after the initial knockout. Mice were then given subcuta-neous injections of luciferase expressing IVC-B16/F10 Melanoma cells in DMEM. The mice were anesthetized with isofluorane for each injec- tion contained 1x106 cancer cells. The mice were then monitored for tumor growth, and once developed the tumors were measured
every two to three days. Tumor vol-ume was extrapolated using the formula length times width squared time 0.532. When any diameter of the tumor exceeded 1.5 cm in length, the mouse was sacrificed.
Western Blotting. Western Blot analysis was conducted on brain, heart, kidney, liver, lung, and spleen tissue harvested from the sacrificed mice. Tissue was homogenized in 87%lysis buffer with 10% triton, 2% 0.5M NaVO4, 1% 100XNEM, and 1 protease inhibitor tablet. These tissue sam-ples were then run on a 7.5% SDS PAGE gel and transferred to nitrocellu-lose membrane, which was cut into strips according to the range marker. The strips
were blocked in 5% milk in Tris Buffered Saline with Tween-20 (TBST) for 45 minutes. The strips were then incubated in
primary antibodies rabbit α epsin diluted 1:4000 in TBST and mouse α GapdH di- luted 1:5000 in TBST overnight at 4ºC. The strips were washed in TBST and incubated in goat α rabbit IgG and goat α mouse IgG both diluted 1:5000 in TBST for one hour at room temperature. The blots were then developed.
Hypoxia Staining. Sections of tumor from the sacrificed mice were imbedded in paraffin for later microscopy. These slides were later deparaf-finized, and antigen retrieval was conducted in 0.05% Pronase in PBS at 40ºC. Primary antibody for this stain was Hy-poxyprobe-1MAb1 diluted 1:50 in PBS, and secondary was Biotin- conjugated F (ab’)2 rabbit α mouse IgG diluted 1:500 in Phosphate Buffered Saline (PBS). DAB kit was added, and the slide was
counter-stained with Hematoxylin. These slides were mounted with Permafluor and viewed on a light micro-scope with a 4X objective. Quantitative analysis of hypoxia was conducted by comparing color threshold masks of tumor samples with and without primary antibody. The masks were created in ImageJ, and ana-lyzed to yield the percent of the sample area that displayed the stain color associated with hypoxia. The difference between the unstained and the stained sections repre-sented the total percentage of cells suffering hypoxia in the sample.
TUNEL Staining. Sections of tumor from the sacrificed mice were imbedded in paraffin for later microscopy. These slides were later deparaf-finized, and antigen retrieval was conducted in Citric Acid Solution with Tween-20 brought to a boil. Slides were
Figure 2. Western Blot Analysis shows the relative protein amounts of epsin and GapdH in VE-Cad knockout tissue. GapdH serves as a control with equal protein amounts, and epsin 1 is noticeably knocked-down in KO tissue, particularly liver and spleen tissues.
Figure 1. Wild Type Mice in both injection batches developed larger tumors more quickly than VE-Cad or Tamoxifen knockout mice.
incubated in TUNEL solution con-sisting of 45 μL Label Solution and 5μL enzyme for one hour in darkness, and slides were mounted in Permafluor and viewed under a confocal micro-scope using a 10X and 20X objective. Quantitative analysis was conducted as described for the Hypoxia slides.
CD-31 Staining. Mice were injected with FITC-Dextran 30 minutes prior to sacrific- ing. Tumor samples were harvested and preserved in O.C.T embedding medium and flash frozen in liquid nitrogen. Samples were cut 30 microns thick and fixed on slides. They were then blocked in PBS with 3% Bovine Serum Albumin (BSA), 3% donkey serum and 0.3% triton. Primary antibody was CD-31 di-luted 1:4 in PBS with triton and 1% BSA. Secondary antibody was donkey α rat AF594 in PBS. Slides were fixed in 4% paraformalde-hyde and mounted with Permafluor and viewed under a confocal
fluores-cent microscope using the 20X and 40X oil immer-sion objectives.
RESULTS The tumors in the knockout mice tended
to develop much more quickly than those in the wild type mice. Figure 1 shows the growth of total tumor volume of global tamoxifen, VE-Cad and wild type mice. The mice were injected in two batches approximately two weeks apart. After almost two months, the global tamoxifen knockout mice of the first batch have yet to develop even one tu-mor. All of the other mice in this group have been sacrificed with large tu-mors. Although the VE-Cad mice did develop tu-mors significantly before the tamoxifen knockout mice, they also showed signs of tumors well after the wild type mice. This confirms our hypothesis that knockout mice would experience inhibited tumor growth,
and the rest of the experiment was designed to provide insight into what mechanisms caused this inhibition.
Western Blotting of the VE-Cad knockout mice confirmed that the knock-out of epsin is not uniform throughout body tissue. In our mice, the ep-sin knockout was most obvious in the brain and liver (Fig. 2). This confirms the physiological difference between the VE- Cad and Global Tamoxifen knock-out mice and provides a likely explanation for the difference between global and endothelial cell spe-cific knockout tumor growth.
The TUNEL and Hypoxia staining procedures highlight cell death and lack of oxygen, respec-tively. Our Hypoxia stain- ing demonstrated a significant increase in cells lacking oxygen in knockout mice. The TUNEL stain-ing, similarly, highlighted large areas of dead cells along the outer edges of knockout tumors, while wild type tumors exhibited only limited and scattered cell death. Finally, CD-31 staining confirmed the de-regulation of angiogenesis. The knockout tumor sam-ples stained with CD- 31 exhibited much thicker and disorganized vasculature, while the vessels in the wild type samples tended to be organized and evenly spaced. This confirms our hypothesis that epsin is needed to keep vasculature growth regulated.
DISCUSSION
Our data confirmed that epsin knockouts inhibit malignant melanoma and glioma tumor growth. When monitoring the mice, we discovered that the wild type mice devel-oped tumors much more quickly than the knockout mice (Fig.1). The mice were generally sacrificed when the longest tumor diameter exceeded 1.5cm, but in some cases a knock-out tumor sample was needed to compare to wild type tumor data, and a knockout mouse had to be sacrificed before reaching the maximum allowed tu- mor size. This influenced our final tumor volume measures, but the slowed growth pattern of tumors in mice not expressing ep- sin remained obvious. The tumor growth in mice with a global tamoxifen knock-out of epsin was slower than the VE-Cad knockout tumor growth as well as the wild type tumor growth. This is likely re-lated to the Western Blotting results, which confirmed that VE- Cad mice did not experience a total knockout of epsin in all major organs. Tumor growth seems to correlate directly with the extent of the epsin knockout in these experiments.
Interestingly, none of the global tamoxifen knockout mice from the first batch of mice
Figure 3. Tumor vasculature in knockout (KO) mice is denser, but less organized than that of wild type (WT) mice as depicted by CD-31 fluorescent staining.
Figure 4. TUNEL staining causes dead cells to glow green under a fluorescent microscope. Tumors in knockout mice show greater rates of cell death than those in wild type mice.
receiv-ing tumors developed a tumor in the first two months of monitoring. The cells implanted in these mice were from the same culture as the cells implanted in the VE-Cad and wild type mice, all of which developed tumors large enough to sacrifice the mouse. One of these mice was infected with glioma tumor cells, and the other two were in-jected with melanoma cells. In the second batch of mice, those injected with tumor cells on June 9, one of the tamoxifen knockout mice developed a tumor in the first 40 days compared to two VE-Cad knockout mice and three wild type mice. We were surprised by the extent of the tumor inhi-bition in the knockout mice, but the result is strong evidence of the role epsin plays in tumor angiogenesis.
Although the death by suffocation of tumors with very dense vascula-ture may seem counterintuitive, the re-sults of our experiment reveal that without epsin angiogenesis can, in fact, perpetuate to a point that the new vessels choke, rather than oxygenate the tumor. CD-31 staining re-veals that the knockout mice actually developed more, and sometimes larger, blood vessels, but the FITC-Dextran in-jected into the mice was unable to penetrate these new vessels (Fig. 3). Blood is likewise unable to reach the tumor through these vessels; they are useless to the tu-mor.
Staining procedures revealed that cell death, particularly along the edges of the tumors, in-creased dramatically in knockout mice (Fig. 4). These cells at the edges of the tumors also showed signs of hypoxia, or lack
of oxygenation. These re-sults are indicative of the disruptive vasculature caused by the deregulation of angiogenesis. The dis-ordered growth of blood vessels to the tumor causes a lack of oxygen, particularly around the edges of the tumor, where the cells eventually die. This reduces the possibil-ity of tumor growth and metastasis.
Overall, the results of our experiment suggest that the amount of epsin in an organism has a defi-nite impact on the growth of tumor cells in that or-ganism. Any amount of epsin deletion can signifi- cantly slow tumor progression, and complete deletions of epsin provide striking results. The corre-lation between epsin, Notch signaling, and an-giogenesis is an important one in understanding the progression of one of the world’s most famous killers.
ACKNOWLEDGMENTS This project could not have been completed
without the support of the Oklahoma Medical Re-search Foundation, and funding from the Puter-baugh Foundation. Dr. Hong Chen has been an incredible resource, and I
am extremely grateful to her for the chance to par-ticipate in her research this summer. The mem-bers of Dr. Chen’s lab have been helpful and pleasant to work with. I would especially like to thank Dr. Satish Pasula, Cindy Maddera, and John McManus for taking the time to walk me through experiments as well as Laurencia Trujillo and Christine Trahms for sharing this experience with me.
REFERENCES 1. Anna-Karin Olsson, Anna Dimberg, Jo-
han Kreuger and Lena Claesson-Welsh 2006 VEGF receptor sig-naling−in control of vascular function. Na-ture Reviews / Mole-cular Cell Biology, VOL. 7, PG. 359.
2. Hong Chen, Silvia Fre. Valdimir I. Slep-nev, Maria R. Capua, Kohji Takei, Margaret H. Butler, Pier Paolo Di Fiore, Pietro De Camilli 1998 Epsin is an EH-domain-binding protein impli- cated in clathrin mediated endocytosis. Nature, vol. 394, pg. 793.
3. Hong Chen, Gevevieve Ko, Ales-san- dra Zatti, Giuseppina Di Gia-como, Lijaun Liu, Elisabetta Raiteri, Ezio Perucco, Chiara Collesi, Wang Min, Caroline Zeiss, Pietro De Camilli, Ot- tavio Cremona. Embryonic arrest at midgestation and disruption of Notch signaling pro-duced by the absence of both epsin 1 and epsin 2 in mice. Proc Natl Acad Sci U S A., 2009 (In Press).
4. Hong Chen and Pie-tro De Camilli. The association of epsin with ubiquit- inated cargo along the endo-cytic path- way is negatively regulated by its inter- action with clathrin. Proc Natl Acad sci U S A, 2004 (In Press).