EXPERT
Expert classes
Lecture 1
Diagnostic classification: Breslow
levels
Measures tumour depth in mm
Skin thickness differs per location.
Diagnostic classification: Clark levels
Correlation between anatomic level of
invasion and mitotic index. More accurate.
Level 1 – 5. Level 4 and 5 are going clearly
deeper; VGP.
Melanoma progression correlates with
ALCAM expression.
o Clark V: 100% positive for ALCAM.
o But it is quite strange that the
clustering molecule ALCAM levels
increase, while the tumour is
invasive.
ALCAM expression: counterproductive event
Release of invasive cells required for metastasis.
Increased ALCAM expression and cell clustering.
Proteases and melanoma progression
Cleavage of receptors, growth factors, adhesion molecules
Extracellular matrix degradation and remodelling.
o The tumour cells need to walk over something, therefore the extracellular matrix is
degraded.
Cleavage of basement membrane.
o Obviously
Matrix metalloproteinase 2 (MMPs) associated with progression and metastasis.
1
, EXPERT
What is the function of ALCAM in tumour progression?
ALCAM structure
Extracellular: D1-D5
TM: transmembrane
D1 can interact with another D1 of an
ALCAM molecule on another cell. D3-D5 will form a dimer to an ALCAM on the same cell.
Member of immunoglobulin superfamily (IgG)
Two V-type IG domains, three C-type domains
Heterophilic (CD6) and homophilic (ALCAM-ALCAM) interactions
ALCAM clustering and network
formation.
These are overexpressed; overexpression
system. Both dN-ALCAM and s-ALCAM
2
, EXPERT
dN-ALCAM
ALCAM and MMP2 activation
SDS-PAGE: separation by size of the proteins.
Zymography is the other way around. 99% SDS-
PAGE with gelatine.
o MMP2 is the enzyme that can cleave the
substrate in the zymography. Gelatin is
the substrate (protein) which gets
cleaved by the MMP2 (gelatinase). The
whole gel is blue (staining of protein
gelatin), except for the places where the
substrate is cleaved by MMP2, you will
see white bands there.
o White bands (below) indicate enzyme activity and cleavage of the substrate.
o Collagen is needed to activate the MMP2. This causes the cleavage in the right
picture by BLM. dN-ALCAM have no network and no ALCAM-ALCAM interactions,
there is also no cleavage of MMP2 visible. This indicates that ALCAM-ALCAM
interactions activate the enzyme MMP2 to cleave the substrate (gelatine) leading to
white bands on the zymography gel.
o ALCAM-ALCAM interactions are important in the activation of MMP2.
3
, EXPERT
Pro-MMP2 is the inactive form of MMP-
2.
Soluble s-ALCAM
Impaired cell-cell interactions. No network formation at all.
Alternative transcript in mice endothelial cells.
Only the NH2 terminal sequence D1; D1 domain is present. This is floating around in the
extracellular matrix.
sALCAM prevents translateral ALCAM membrane recruitment.
4
Expert classes
Lecture 1
Diagnostic classification: Breslow
levels
Measures tumour depth in mm
Skin thickness differs per location.
Diagnostic classification: Clark levels
Correlation between anatomic level of
invasion and mitotic index. More accurate.
Level 1 – 5. Level 4 and 5 are going clearly
deeper; VGP.
Melanoma progression correlates with
ALCAM expression.
o Clark V: 100% positive for ALCAM.
o But it is quite strange that the
clustering molecule ALCAM levels
increase, while the tumour is
invasive.
ALCAM expression: counterproductive event
Release of invasive cells required for metastasis.
Increased ALCAM expression and cell clustering.
Proteases and melanoma progression
Cleavage of receptors, growth factors, adhesion molecules
Extracellular matrix degradation and remodelling.
o The tumour cells need to walk over something, therefore the extracellular matrix is
degraded.
Cleavage of basement membrane.
o Obviously
Matrix metalloproteinase 2 (MMPs) associated with progression and metastasis.
1
, EXPERT
What is the function of ALCAM in tumour progression?
ALCAM structure
Extracellular: D1-D5
TM: transmembrane
D1 can interact with another D1 of an
ALCAM molecule on another cell. D3-D5 will form a dimer to an ALCAM on the same cell.
Member of immunoglobulin superfamily (IgG)
Two V-type IG domains, three C-type domains
Heterophilic (CD6) and homophilic (ALCAM-ALCAM) interactions
ALCAM clustering and network
formation.
These are overexpressed; overexpression
system. Both dN-ALCAM and s-ALCAM
2
, EXPERT
dN-ALCAM
ALCAM and MMP2 activation
SDS-PAGE: separation by size of the proteins.
Zymography is the other way around. 99% SDS-
PAGE with gelatine.
o MMP2 is the enzyme that can cleave the
substrate in the zymography. Gelatin is
the substrate (protein) which gets
cleaved by the MMP2 (gelatinase). The
whole gel is blue (staining of protein
gelatin), except for the places where the
substrate is cleaved by MMP2, you will
see white bands there.
o White bands (below) indicate enzyme activity and cleavage of the substrate.
o Collagen is needed to activate the MMP2. This causes the cleavage in the right
picture by BLM. dN-ALCAM have no network and no ALCAM-ALCAM interactions,
there is also no cleavage of MMP2 visible. This indicates that ALCAM-ALCAM
interactions activate the enzyme MMP2 to cleave the substrate (gelatine) leading to
white bands on the zymography gel.
o ALCAM-ALCAM interactions are important in the activation of MMP2.
3
, EXPERT
Pro-MMP2 is the inactive form of MMP-
2.
Soluble s-ALCAM
Impaired cell-cell interactions. No network formation at all.
Alternative transcript in mice endothelial cells.
Only the NH2 terminal sequence D1; D1 domain is present. This is floating around in the
extracellular matrix.
sALCAM prevents translateral ALCAM membrane recruitment.
4
