Status of CDK2, CDK1 and CDK5 expression in normal skin, nevus, and melanoma tissues.
To determine the status of CDK2, CDK1 and CDK5 expression in the melanoma progression pathway, tissue samples representing normal skin, benign nevi, atypical nevi, which are the precursors and risk markers of melanoma, melanoma in situ, which although noninvasive is the first stage of melanoma development, VGP melanoma and subcutaneous and visceral MGP melanoma, were probed with antibody to human CDK2, CDK1 and CDK5. The results of this immunohistochemistry analysis () revealed no expression of CDK2, CDK1, or CDK5 in junctional melanocytes of normal skin. Similarly, none of these three CDKs was expressed in nevocytes, which are the clusters of melanocytes in benign and atypical nevi, and they were not expressed in melanoma in situ cells. In contrast, VGP melanomas, and to an even greater extent, MGP melanomas, demonstrated strong expression of CDK2, CDK1 and CDK5. Given the different histologic stages, at least two and in most cases, seven representative tissue samples were probed with the different CDK antibodies. All tissue specimens revealed a similar pattern and status of CDK2, CDK1 and CDK5 expression as the tissue samples depicted in .
Figure 1 Expression of CDK2, CDK1, and CDK5 in the melanoma progression pathway. Five-µm sections, prepared from cryopreserved tissue specimens representing normal skin, benign nevus, atypical nevus, melanoma in situ and VGP and MGP melanoma were probed (more ...)
Expression of cyclin-dependent kinases in melanoma cells, melanocytes, and skin fibroblasts.
a 33 kD nuclear protein, is inactive until complexed with cyclin E at G1
and thereafter with cyclin A for S-phase progression.12,13
Activation of the cyclin/Cdk2 complex requires dephosphorylation of CDK2 at threonine 14 (Thr-14) and tyrosine 15 (Tyr-15) and phosphorylation at threonine (Thr-160).
To obtain information as to whether in cells representing advanced melanoma, CDK2 is present predominantly in its monomeric form or in a complex, we probed total cell lysates, prepared from melanoma cells derived from five different MGP melanomas, with an antibody to total CDK2 and an antibody that recognizes phospho(p)-CDK2 (Thr-14). The data of this immunoblot analysis revealed that all five non-synchronized MGP melanoma cell lines expressed two proteins of approximately 50 and 70 kD, which may represent complexed CDK2 (, lanes c–g). Two of these five MGP melanoma cell lines, WM1158 (, lane c) and WM852 (, lane d), did not show expression of the 33 kD monomeric form of CDK2, but did show strong expression of pCDK2 (Thr-14), while the other three cell lines, WM266-4 (, lane e), SK-MEL-28 (, lane f), WM983-B (, lane g), revealed the opposite. Since the results of the immunohistochemistry analysis () had demonstrated stronger expression of CDK2 in MGP compared with VGP melanoma tissues, we also performed an immunoblot analysis of two cell lines that were established from a VGP and an MGP melanoma of a same patient. This side-by-side comparison showed that unlike the WM983-B MGP melanoma cells (, lane b), the WM983-A VGP melanoma cells (, lane a) did not express the 33 kD monomeric form of CDK2, and the intensity of the 50 and 70 kD proteins was slightly less.
Figure 2 Expression of CDK2, pCDK2, CDK9, CDK5 and CDK1 in VGP and MGP melanoma cell lines, and in human neonatal melanocytes and skin fibroblasts. (A) Immunoblot analysis of CDK2, pCDK2, and CDK9 expression in the VGP melanoma cell line WM983-A (a), in MGP melanoma (more ...)
Also depicted in are total cell lysates of human neonatal melanocytes (, lane h), which showed expression of the 70 kD protein, the monomeric 33 kD CDK2 protein and CDK2 phosphorylated at Thr-14. Not present in these melanocytes was the 50 kD protein. In contrast, human neonatal skin fibroblasts demonstrated in addition to the 70 kD protein, strong expression of the 50 kD protein (, lane wi) and the presence of phosphorylated CDK2. The finding that, albeit at low levels, short-term cultures of human neonatal melanocytes express CDK2, but not as shown in , melanocytes residing in the epidermal/dermal junction of normal human skin tissue, may be attributable to the fact that in order for human neonatal melanocytes to proliferate for a few passages in vitro before they undergo senescence, their growth medium has to be supplemented not only with the tumor promoter, TPA, but also with considerably high amounts of basic fibroblast growth factor (bFGF).14
Because of our subsequently performed molecular targeting studies, described below, which involved the use of the small-molecule inhibitor, SCH 727965, which in addition to CDK2, inhibits CDK5, CDK1 and CDK9, we performed as depicted in and , additional immunoblot analyses to determine the status of expression of these latter three CDKs in the different melanoma cell lines. The results demonstrated that all five MGP melanoma cell lines (, lanes c–g) as well as human neonatal melanocytes (, lane h), strongly expressed the 42 kD isoform of CDK9, which shares 47% homology with other CDKs, complexes with cyclin T variants, and functions primarily in the elongation of RNA transcripts.15–17
Comparatively weak was the expression of CDK9 in short-term cultures of human neonatal skin fibroblasts (, lane i).
Unlike CDK2, CDK1 or the other known CDKs, CDK518,19
does not require for its activation phosphorylation on the T loop, and it is not a checkpoint kinase, but functions as a regulatory kinase in important neuronal processes, including brain development and neuronal stress responses (for review, see reference 20
). Yet, like the tissue specimens representing advanced melanoma (see ), every one of the five MGP melanoma cell lines expressed clearly detectable levels of CDK5 (, lanes a–e).
CDK1 complexed with cyclin A in G2 and cyclin B in M phase, is the only CDK that can drive mammalian cells through the entire cell cycle (for review, see reference 21
). Isoform 1 of CDK1 codes for a protein of 34 kD, which was strongly expressed in 2/5 MGP melanoma cell lines (, lane a and e
), whereas all five cell lines revealed strong expression of a protein of about 50 kD, which may represent complexed CDK1.
CDK inhibitor treatment of melanoma cells impairs their CDK activity and phosphorylation of the retinoblastoma protein.
Using in vitro kinase assays, it was previously determined22
that the small-molecule inhibitor, SCH 727965, inhibits CDK2, CDK5, CDK1 and CDK9 activity with IC50
values of 1, 1, 3 and 4 nmol/L, respectively. Given the high expression of CDK2 and the three other CDKs in tissues and cell lines representing advanced melanoma, we projected that it might require a relatively high dose of SCH 727965 to block the function of these CDKs in melanoma cell lines. Thus, given the first experiment, we treated WM1158 MGP melanoma cells for 24, 48 and 72 hr with 1 µM of this small-molecule inhibitor. Total cell lysates from these CDK inhibitor-treated cells were then probed with antibody to pCDK2 (Thr-14), total CDK9, or the 110 kD retinoblastoma protein phosphorylated at Thre-821, the site that is phosphorylated by complexed CDK2/cyclin A and CDK2/ cyclin E. As depicted in , compared with WM1158 MGP melanoma cells that had received only the small-molecule inhibitory delivery vehicle, Dimethyl Sulfoxide (DMSO), WM1158 cells that had been incubated in the presence of SCH 727965 revealed that as early as 24 hr post treatment, CDK2 activity and CDK9 expression were impaired and the retinoblastoma protein was no longer phosphorylated.
Figure 3 Impact of CDK inhibitor treatment of melanoma cells upon the expression of pCDK2, CDK9 and pRb. (A) pCDK2 (Thr-14), CDK9 and pRb (THR-821) immunoblot analysis of WM1158 MGP melanoma cells that received only DMSO (−) or were treated with 1 µM (more ...)
Immunofluorescence analysis of WM1158 MGP melanoma cells, treated for 24 hr with 1 µM of the small-molecule inhibitor, also demonstrated significantly less pCDK2 expression compared with WM1158 MGP melanoma that had received only DMSO (). Likewise, immunofluorescence analysis of CDK inhibitor and DMSO-treated WM1158 cells performed with an antibody to (total) CDK2 or CDK9, showed noticeably less CDK2 as well as CDK9 expression in the small-molecule inhibitor treated melanoma cells (data not shown).
CDK inhibitor treatment blocks melanoma cell proliferation and impairs melanoma cell cycle progression.
To determine whether treatment with the SCH 727965 small-molecule agent, which as previously reported is active against a broad spectrum of human cancer cell lines,22
would have an impact on melanoma cell proliferation, we performed an experiment wherein MGP melanoma cells were incubated in the presence of three increasing doses of this CDK inhibitor. Although the results demonstrated that over a period of 96 hr, all three doses impaired melanoma cell proliferation (), the lowest dose of 0.5 µM was not as effective as the two higher doses of 1 µM and 5 µM, and there was not a substantial difference between the latter two. The fact that treatment with the inhibitor severely interfered with the proliferation of melanoma cells became even more apparent in the context of a second experiment (), wherein we compared the proliferation of CDK inhibitor to DMSO-treated WM1158 and likewise, WM852 (data not shown) MGP melanoma cells.
Figure 4 CDK inhibitor treatment of melanoma cells leads to inhibition of melanoma cell proliferation and dysregulation of melanoma cell cycle progression. (A) Proliferation of MGP melanoma cells (WM1158) incubated in the presence of 0.5 µM, 1 µM, (more ...)
Interestingly, but not entirely unexpected, it was not only the cells' proliferation that was majorly impaired upon treatment with the CDK inhibitor but also their morphology. As depicted in , within 24 hours following addition of the inhibitor, and even more pronounced thereafter, the cells severed their cell-cell contacts while at the same time, a limited but noticeable number of cells that continued to adhere to the surface of the Petri dishes, became overtly round in shape and larger in size—an indication that these cells were unable to progress through the entire cell cycle. To obtain experimental evidence in support of this observation, WM1158 MGP melanoma cells that had been treated with 0.5 µM or 1 µM of the CDK inhibitor, or had received only DMSO, were labeled with Propidium Iodide and analyzed by flow cytometry. In comparison with the DMSO-treated melanoma cells, the CDK inhibitor-treated melanoma cells demonstrated an increased accumulation in S phase and a significantly reduced presence in G2/M phase (), suggesting that their DNA replication and subsequently, cell division were impaired.
CDK inhibitor treatment leads rapidly to massive apoptosis of melanoma cells.
One of the prominent biological characteristics of VGP and MGP melanomas is their extreme resistance to apoptosis. Thus, it was surprising that in addition to being impaired in their proliferation and cell cycle progression, the CDK inhibitor-treated melanoma cells displayed major signs indicative of apoptosis. Following TUNEL as well as Annexin V staining, immunofluorescence analysis revealed significant numbers of apoptotic cells among WM1158 MGP melanoma cells that had been treated with 1 µM of the CDK inhibitor for 48 and 72 hours (), whereas WM1158 melanoma cells that had received only DMSO showed no TUNEL and very little Annexin V staining ().
Figure 5 Inhibiting the function of CDKs in melanoma cells leads to melanoma cell apoptosis. (A) WM1158 MGP melanoma cells, treated with 1 µM of the CDK inhibitor (CDKI) for 48 or 72 hr, were analyzed by immunofluorescence-based TUNEL and likewise, Annexin (more ...)
Furthermore, c-PARP immunoblot analysis documented that WM1158 and likewise, WM852 (data not shown) MGP melanoma cells treated with the CDK inhibitor at a dose of 1 µM or higher, underwent apoptosis within 24 hours following addition of the inhibitor as reflected by cleavage of PARP to cPARP (). The data of these experiments were further supported by the results of an Annexin V/Propidium Iodide-based flow cytometry analysis (), which showed that regardless of whether the CDK inhibitor was present for 24, 48, or 72 hours, it was not stress-induced necrosis but apoptosis that led to melanoma cell death and the cells' inability to resume growth after they had been rinsed and resuspended in medium not containing the inhibitor (data not shown).
In vivo and ex vivo analysis of CDK inhibitor-treated human melanoma xenografts.
Given the extreme resistance of advanced melanoma to standard regimens of treatment, and the fact that, to date, limited information is available regarding genes that might be suitable targets for molecular therapy of advanced melanoma, we next undertook a series of preclinical studies to determine whether CDK inhibitor treatment would have efficacy for human MGP melanoma xenografts grown as subcutaneous tumors in nude mice.
Specifically, these in vivo studies focused upon systemic, intraperitoneal (i.p.) treatment of nude mice bearing WM983-B MGP human melanoma xenografts with 40 mg/kg of the CDK inhibitor first administered on the day (Day 1) a tumor had reached 5 mm in any dimension. Thereafter, the tumor-bearing animals were injected three more times with the same and well-tolerated 40 mg/kg dose of the inhibitor, which was below the as previously in a human lung adenocarcinoma (A549) xenograft model determined maximally tolerated dose of 87 mg/kg.22
Following the last injection on Day 10, tumor sizes were measured for an additional period of two weeks at which point the animals were sacrificed because the tumors in the DMSO control arm had reached the maximum allowable size. A second experimental arm was comprised of WM983-B MGP melanoma xenograft-bearing nude mice that using the same dose of 40 mg/kg, were given i.p. injections of the CDK inhibitor every third or fourth day until Day 17. The results of this series of preclinical studies () revealed that compared with the tumors in the nude mice that had received i.p. injections of only DMSO, the tumors in the animals that had been treated a total of four times with the CDK inhibitor grew noticeably slower as did the tumors in the animals that had received the inhibitor six times.
Figure 6 CDK inhibitor treatment of human melanoma xenograft-bearing nude mice. (A) Representative, individual examples of the growth rate of subcutaneous WM983-B MGP melanoma xenografts in nude mice that received the first i.p. injection of the CDK inhibitor (more ...)
Since it is unlikely that a small-molecule inhibitor, regardless of its molecular target, when administered as a single agent will ever be effective to the extent that it will be a cure for patients with advanced melanoma, we next determined whether a combination treatment would further enhance the impact of the CDK inhibitor on the growth of human MGP melanoma xenografts. Thus, we administered the CDK inhibitor (40 mg/kg) in combination with 10 mg/kg of the cytotoxic drug Paclitaxel, frequently used as a combination agent in melanoma-based clinical trials. Using a schedule of i.p. injections that spanned Day 1–10, Paclitaxel was administered 24 hr following each injection of the CDK inhibitor. As shown in , compared with the growth rate of the tumors in the nude mice that had been treated with only the inhibitor, the growth of the tumors in the animals that had received the combination treatment was further reduced.
Given the results of these in vivo molecular targeting studies, we next determined the extent to which the systemic i.p. treatment with the small-molecule CDK inhibitor when administered alone or in combination with Paclitaxel had reached and blocked its molecular targets in the MGP melanoma xenografts. For this purpose, we probed tissue sections from several of the resected tumors with antibody to (total) CDK2 as well as CDK5.
As documented in , tumor sections prepared from WM983-B MGP human melanoma xenografts of nude mice that had been treated with only the CDK inhibitor, or a combination of CDK inhibitor and Paclitaxel, exhibited noticeably less CDK2 and likewise, CDK5 expression than the MGP melanoma xenografts from the animals that had been given i.p. injections of only DMSO, or serving as another control arm for the CDKI/Paclitaxel treatment, only Paclitaxel. Furthermore, and similar to the CDK inhibitor-induced morphological cell changes, we had observed in the setting of our in vitro studies (), fluorescent DAPI staining of these different MGP melanoma xenograft tissue sections () revealed that the cells in the CDK inhibitor and even more so the CDK inhibitor/Paclitaxel-treated tumor xenografts had noticeably enlarged nuclei when compared with the nuclei of cells in tumors that represented the DMSO or Paclitaxel control arm. We also probed tissue sections, prepared from the different tumors with antibody to human pRb and pCDK2. However, neither antibody gave consistent and reproducible staining in the case of the DMSO and likewise, Paclitaxel control tumors (data not shown).