Establishment and Characterization of Melanoma Cells Expressing DPPIV.
To define a possible functional role of DPPIV in melanocytic cells, we established melanoma cells that expressed DPPIV in an inducible manner using tetracycline-inducible vectors. Three human melanoma cell lines, MEL-22a, SK-MEL-28, and SK-MEL-29, derived from metastatic lesions of different patients, were selected for study. These melanoma cell lines are representative of more than 150 melanoma cell lines that we have tested that do not express detectable DPPIV glycoprotein (reference 2 and our unpublished data). In addition, the growth and differentiation of these three melanoma lines have been well characterized 1718
. These cell lines represent different stages of melanocyte/melanoma differentiation 17
. They are either completely nonpigmented with a phenotype that corresponds to an immature stage of melanocyte differentiation (MEL-22a) or minimally pigmented with a phenotype of an intermediate stage of melanocyte differentiation (SK-MEL-28 and SK-MEL-29; reference 17 and 18; ).
Characteristics of Human Melanoma Cells Expressing Wild-Type and Mutant DPPIV
Because we had trouble isolating stable transfectants expressing DPPIV using constitutive vectors, we used an inducible vector system. Melanoma cells were cotransfected with a neomycin-resistant regulator plasmid and tetracycline-inducible vector carrying: (a) the full length wild-type (wt)DPPIV cDNA, (b) a mutant (mut)DPPIV having minimal serine protease activity (substitution of alanine for serine at codon 630 altering the catalytic domain), or (c) a control empty vector 16
. Multiple clones that expressed each cDNA construct were isolated for each cell line ( and ). Transfected clones expressing empty vector and mutDPPIV were always relatively easy to derive, and at least three clones were established for each melanoma cell line. Transfected clones expressing wtDPPIV were more difficult to establish, suggesting that DPPIV expression affected cell survival or growth. Despite this difficulty, seven different clones expressing low, medium, and high levels of DPPIV were selected from MEL-22a transfectants, and two clones each were isolated for SK-MEL-28 and SK-MEL-29.
Range of DPPIV Enzyme Activity
DPPIV expression was assessed by three methods: (a) immunofluorescence staining, (b) immunoprecipitation from metabolically labeled cells, and (c) enzymatic activity. shows DPPIV expression in representative clones of MEL-22a transfectants, with or without induction by dox. DPPIV cell surface expression was substantially induced by dox ( A). Melanoma cells transfected with wtDPPIV and mutDPPIV expressed the expected 110–120-kD glycoprotein, showing that both the wild-type and mutant polypeptides were processed and expressed appropriately ( B). Furthermore, these results showed that mutDPPIV was stable and expressed at levels comparable to those of wtDPPI. A weak 110-kD band was detected in parental cells and cells transfected with empty vector upon long exposure of autoradiographs ( B), suggesting that melanoma cells can express very low levels of endogenous DPPIV.
Figure 1 Expression of DPPIV in transfected melanoma cells. (A) Immunofluorescence microscopy showing expression of DPPIV. MEL-22a cells were cultured in the presence or absence of dox (2 μg/ml) for 48 h and stained with S27 mAb against DPPIV. Groups include (more ...)
Transfected clones and parental melanoma cells were assessed for DPPIV enzymatic activity ( and C). Parental and vector control–transfected cells expressed ≤30 pM/min/μg protein of DPPIV activity, which we believe represents very low endogenous DPPIV activity. In the absence of dox, DPPIV activity in DPPIV-transfected melanoma cells was ≤60 pM/min/μg protein. Peptidase activity of melanoma cells induced to express high levels of wtDPPIV in the presence of dox was 220–310 pM/min/μg protein ( and C). This level was comparable to that of melanocytes (300–350 pM/min/μg protein; range from three distinct assays; C). Despite high expression of mutDPPIV protein ( and ), melanoma cells expressing mutDPPIV exhibited low levels of enzyme activity even in the presence of dox (≤60 pM/min/μg protein; and C). Transfected MEL-22a clones were isolated that expressed high (hi), medium (med), and low DPPIV activity for more detailed studies to compare phenotype and level of DPPIV expression ( and ).
In summary, levels of DPPIV expression were consistent across the three assays, showing that steady-state level of protein expression corresponded to wtDPPIV enzymatic activity. As expected, there was low DPPIV enzyme activity in cells expressing mutDPPIV. Results of DPPIV activity in transfected melanoma lines SK-MEL-28 and SK-MEL-29 showed levels similar to those of MEL-22a (). These results showed that: (a) the maximum level of DPPIV activity in transfected melanoma cells did not exceed levels expressed by cultured normal melanocytes (either normalized to protein concentration or when calculated on a per-cell basis), (b) dox induced DPPIV expression fivefold or more, and (c) mutDPPIV expressed minimal or no enzyme activity.
Inhibition of Tumorigenicity by Expression of DPPIV.
Tumorigenicity of melanoma cells expressing wtDPPIV or mutDPPIV was compared with that of control melanoma cells. Nude mice were injected subcutaneously with transfected and control MEL-22a or SK-MEL-29 melanoma cells (parental SK-MEL-28 melanoma cells do not form tumors in immune-compromised mice). Parental and control vector melanoma cells formed progressive tumors in all mice. and shows results from two different experiments for MEL-22a, and C shows results for SK-MEL-29. Tumorigenicity was essentially ablated in MEL-22a cells when DPPIV was induced to levels expressed by normal melanocytes (wtDPPIVhi). Mice showed no progression of tumors over 100 d ( and ), although viable tumor cells remained after 100 d (data not shown). Similar results were observed with SK-MEL-29 expressing wtDPPIV ( C). Tumor growth was also reduced in melanoma cells expressing medium levels of DPPIV, although not as profoundly as for high levels of DPPIV ( and ). Transfected MEL-22a melanoma cells expressing low levels of DPPIV, either in the absence of induction of DPPIV (wtDPPIVhi [−dox], A) or constitutively (DPPIVlow [+dox], B), showed slightly reduced tumor growth, perhaps due to either low levels of DPPIV activity or recruitment of FAPα, which forms a heterodimer with DPPIV (as discussed below). Melanoma cells expressing high levels of mutDPPIV formed tumors at variable rates, with some mice showing inhibition of tumor growth (note error bars in and for mutDPPIV). These results were consistent with a require-ment of DPPIV serine peptidase activity for complete inhibition of tumorigenicity. However, inconsistent inhibition of tumorigenicity in melanoma cells expressing mutDPPIV suggested that some effects of DPPIV on in vivo tumor growth were possibly independent of DPPIV serine peptidase activity.
Figure 2 Effects of DPPIV expression on tumorigenicity. (A) Expression of wtDPPIV was associated with inhibition of tumor growth of melanoma cells MEL-22a in nude mice. Six different sets of nude mice (BALB/C nu/nu, n = 5–6 for each group) were challenged (more ...)
Inhibition of Anchorage-independent Growth by Expression of DPPIV.
Another characteristic of malignant cells is anchorage-independent growth. Expression of DPPIV led to a marked decrease in the ability of MEL-22a melanoma cells to grow in soft agar. DPPIV expression inhibited colony-forming ability by ~75% in MEL-22a cells, with little inhibition of MEL-22a cells expressing mutDPPIV compared with parental and vector control cells (). Thus, serine peptidase activity was required to decrease anchorage-independent growth.
Figure 3 Anchorage-independent growth in soft agar. MEL-22a cells described in A were cultured in the absence (−) or presence (+) of dox for 48 h, and 5,000 viable cells were plated in agar in triplicate as described in Materials and Methods. Results (more ...)
Phenotypic Changes of Melanoma Cells Expressing DPPIV.
Marked morphological changes were observed in melanoma cells expressing wtDPPIV ( and ). Parental MEL-22a melanoma cells and cells transfected with control vector or mutDPPIV were a disorganized array of epithelioid, polygonal, and short bipolar spindle-shaped cells and grew in piled colonies without any apparent organization ( A). Cells expressing medium or high levels of wtDPPIV were consistently long bipolar spindle shaped, with organized growth behavior and sheet-like appearance, suggesting organization by cell–cell contact ( A). SK-MEL-28 and SK-MEL-29 cells also changed morphology from the long spindle shape of parental-, control vector–, and mutDPPIV-transfected cells to a more mature polydendritic shape of cells expressing wtDPPIV (). The polydendritic shape is characteristic of well-differentiated melanoma cells 1718
Figure 4 Phenotypic changes associated with DPPIV expression. (A) Morphology of MEL-22a clones. Untransfected (panel 1) and control vector–transfected cells (panel 2) showed short spindle-shaped and polygonal morphology and grew in unorganized clusters. (more ...)
We have previously shown that MEL-22a cells have a block in differentiation associated with a nonpigmented, immature melanocytic phenotype 18
. Five MEL-22a clones expressing medium and high levels of DPPIV were pigmented when grown to confluence ( B and ). Three clones expressing mutDPPIV had no pigment. None of the six clones with control vector nor parental cells were pigmented ( B and ). Differentiation of melanocytic cells is characterized not only by appearance of pigmentation but by expression of melanosome membrane glycoproteins involved in melanin metabolism. The best characterized glycoproteins are members of the tyrosinase family, including tyrosinase and tyrosinase-related proteins (TRP). Expression of wtDPPIV, but not mutDPPIV, correlated with a markedly increased expression of human tyrosinase ( C). Expression of wtDPPIV (but not mutDPPIV) was also associated with de novo expression of the brown
locus protein, gp75TRP-1 23
, measured by indirect immunofluorescence staining (data not shown). Expression of gp75TRP-1
and upregulation of tyrosinase protein occur at a later stage in melanocyte differentiation, confirming that the tyrosinase low/gp75TRP-1
–negative MEL-22a had differentiated. Induction of pigmentation associated with expression of DPPIV was also observed in the two other melanoma cell lines, SK-MEL-28 and -29 (). These observations show that DPPIV expression is associated with a relief of the block in differentiation of melanoma cells.
Growth Characteristics of Melanoma Cells Expressing DPPIV.
Expression of DPPIV did not affect growth of MEL-22a cells during the logarithmic growth phase. The doubling time of cells expressing high wtDPPIV, mutDPPIV, and control vectors was 36–38 h and was exactly the same as for parental MEL-22a in culture media containing serum (36 h). However, melanoma cells expressing medium and high levels of wtDPPIV had a much longer lag period after plating before they entered the logarithmic growth phase (4–5 d) compared with parental cells and melanoma cells expressing mutDPPIV and control vectors (1–2 d). Also, growth of wtDPPIV cells was inhibited when cells reached a confluent state, whereas parental melanoma cells and cells expressing mutDPPIV and control vector continued to grow and pile up after reaching confluency ( A). Thus, the total cell number of wtDPPIV cells was decreased by 40% compared with control melanoma cells or mutDPPIV cells on days 10–14 after plating. This was due to the delay before entering logarithmic growth but also perhaps to inhibited growth upon reaching confluency. Thus, wtDPPIV expression did not affect log growth of MEL-22a cells but did slow entry into the rapid growth phase and appeared to induce some level of growth inhibition at cell confluency. The difficulty in initiating growth might explain in part the difficulty in establishing transfected clones of melanoma cells expressing wtDPPIV.
Apoptosis and Block of Cell Cycle in Serum-Free Conditions Induced by DPPIV Expression.
Transformed cells are typically released from dependence on exogenous growth factors for survival during tumor progression 24
. This characteristic applies to melanoma cells, which have been shown to survive and grow in serum-free culture medium without addition of exogenous growth factors, whereas normal melanocytes die over 7–14 d when serum is withdrawn 2526
. We had previously shown that loss of DPPIV expression was associated with acquisition of growth factor independence during in vitro transformation of melanocytes 1413
. This observation demonstrated a correlation between DPPIV expression and a requirement for exogenous growth factors for survival. We investigated this possible link by growing transfected and parental melanoma cells in serum-free conditions.
WtDPPIV, mutDPPIV, and control MEL-22a cells were serum starved with or without induction of DPPIV by dox. Parental and vector control cells grew in serum-free media with only low levels of detectable apoptotic cell death (~2–3% of cells showed DNA fragmentation by TUNEL assay over 15 d) (). A minor population of transfected melanoma cells not induced for wtDPPIV cells demonstrated cell death in serum-free conditions (21% of cells at 15 d; (). However, cells induced to express either wtDPPIV or mutDPPIV with dox showed a marked, progressive loss of cell viability; the proportion of apoptotic cells was 15–18% at day 3, 45–53% at day 8, and 62–78% at day 15 (). Similar results were observed with SK-MEL-29 cells. Only 8% of control vector–transfected cells were apoptotic 8 d after serum withdrawal compared with 52% of cells expressing wtDPPIV.
Apoptosis of MEL-22a Human Melanoma Cells Expressing DPPIV in Serum-free Conditions
The same set of cells was analyzed for cell cycle progression in serum-free conditions (). wtDPPIVhi expression induced a cell cycle arrest at the G0/G1 phase, with 62–76% of the cells present in the G0/G1 stage by day 8 compared with only 5–12% of control vector and parental cells in the G0/G1 (range of percentages from duplicates of two experiments). Interestingly, cell cycle arrest at the G0/G1 stage was detected in 32% of cells expressing mutDPPIV, intermediate between wtDPPIV and melanoma cells not expressing DPPIV. These results with mutDPPIV suggest either that some other function than serine protease activity of DPPIV is involved in apoptosis and cell cycle arrest induced by serum withdrawal or that DPPIV interacts with other molecules that mediate survival and cell cycle effects.
Figure 5 Cell cycle analysis of melanoma cells expressing DPPIV and FAPα. Expression of both wt- and mutDPPIV in MEL-22a cells was associated with changes in cell cycle. Parental MEL-22a cells and cells transfected with control vector, mutDPPIV, and wtDPPIV (more ...)
DPPIV Rescues Expression of FAPα.
FAPα is a potential cell surface serine protease that is coexpressed with DPPIV by melanocytes. Loss of FAPα expression occurs concomitantly with loss of DPPIV expression during in vitro transformation of melanocytes, and expression is also lost in primary and metastatic melanoma cell lines 1415
. DPPIV and FAPα can form heterodimers in addition to homodimers formed by DPPIV 15
. Reexpression of either wt- or mutDPPIV by MEL-22a melanoma cells induced the cell surface expression of FAPα (). The relative level of surface expression of FAPα corresponded to the level of DPPIV expression, irrespective of wild-type or mutant forms. Thus, DPPIV rescued surface expression of FAPα.
Figure 6 Expression of DPPIV and FAPα. Expression of either wt- or mutDPPIV in MEL-22a cells rescued the expression of FAPα. Immunofluorescence staining and flow cytometry analysis of DPPIV-transfected cells was performed using mAbs against DPPIV (more ...)