![Cdk4 down-regulation is a late TGF-B effect in MvlLu and HaCaT cells. Exponentially growing MvlLu and HaCaT cells were treated with 100 pM TGF-f~ for the indicated times. [aSS]Methionine was included in the last hour of incubation with TGF-f3. Cell extracts were precipitated with either Cdk4 antibody (A) or Cdk6 antibody (B) and subjected to SDSPAGE and fluorography.](https://www.researchgate.net/profile/Kornelia-Polyak/publication/15548628/figure/fig1/AS:601586606805045@1520440832901/Cdk4-down-regulation-is-a-late-TGF-B-effect-in-MvlLu-and-HaCaT-cells-Exponentially_Q320.jpg)
![p15 tnk4B mRNA and protein response to TGF-13 in MvlLu cells. (A,B] Northern blot analysis of p15 znk4B mRNA levels with poly(AI+RNA from MvlLu ceils during the indicated proliferative states {A) or after treatment of exponentially growing cells with 100 pM TGF-B for the indicated times (B). The blots were reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH} cDNA probe, a housekeeping gene, to control for RNA loading. (C) Analysis of Cdk6-associated p 15 in MvlLu cells treated with TGF-I3 for the indicated times. Cells were metabolically labeled with [35S]methionine for the last 2 hr of incubation with TGF-B, precipitated with Cdk6 antibody, and subjected to SDS-PAGE and fluorography. An aliquot of the 8 hr extract was immunoprecipitated with preimmune serum as control {lane C). The position of p15 (arrow) is indicated.](https://www.researchgate.net/profile/Kornelia-Polyak/publication/15548628/figure/fig3/AS:601586611003400@1520440833130/p15-tnk4B-mRNA-and-protein-response-to-TGF-13-in-MvlLu-cells-A-B-Northern-blot_Q320.jpg)
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Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta
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G1 progression in mammalian cells requires the activity of the cyclin D-dependent kinases Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. Proliferating Mv1Lu mink lung epithelial cells and human keratinocytes contain high levels of the universal Cdk inhibitor p27Kip1 distributed in complexes with Cdk2, Cdk4, and Cdk6. Addition of the antimitogenic cytokine transforming growth factor-beta (TGF-beta) elevates expression of the Cdk4/6-specific inhibitor p15Ink4B and induces the release of p27 from Cdk4 and Cdk6. In Mv1Lu cells, this release of p27 coincides with increased binding of p27 to Cdk2. Recombinant p15 inhibits p27 binding to Cdk4 in vitro, and p15 overexpression induces the transfer of p27 from Cdk4 to Cdk2 in vivo, suggesting that the release of Cdk4-bound p27 in TGF-beta-treated cells is caused by the surge in p15 levels. In keratinocytes, TGF-beta increases not only p15 but also p21Cip1, which binds to Cdk2. These events correlate with Cdk2 inhibition and cell cycle arrest and occur without a loss of G1 Cdk components. The results suggest that TGF-beta induces G1 arrest in these two epithelial cell types by inhibiting various cyclin-Cdk kinases through the cooperative action of an Ink4 Cdk inhibitor and a Cip/Kip Cdk inhibitor. Subsequent to cell cycle arrest, Cdk2 and Cdk4 levels decline as part of a second set of events that may represent a program of cell adaptation to the quiescent state.
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... The TGFβ family of ligands acts as potent tumor suppressors notably by inducing CDK inhibitors (CDKIs) [38]. Thus, we examined whether TGFβ3 could modulate the expression of the CDK inhibitor p21 in both parental and palbociclib-resistant SUM159 cells. ...
... TGFβ induces the expression of the INK4 family of CDK inhibitors, including p21 CIP1 (p21) [38,59]. It has been shown that CDK4/6 inhibitors, including palbociclib, selectively redistribute p21 from CDK4/cyclin D1 complexes to inhibit CDK2 activity [39]. ...
Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFβ3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFβ3 treatment can overcome this. This study defines TGFβ3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFβ3 to propose a new combinatorial treatment for TNBC.
... Nevertheless, MCF7 cells also responded but rather through the upregulation of cell cycle inhibitors, especially CDKN1A (encoding p21) and CDKN2B (encoding p15). The transcription of both genes has been shown to be upregulated in response to TGFβ [39], [40], [41], [42], [43]. These CDK inhibitors can arrest the cell cycle in the early G1 phase. ...
... These CDK inhibitors can arrest the cell cycle in the early G1 phase. In particular, CDKN2B interacts strongly with CDK4 and CDK6, which are important for cell cycle G1 phase progression, while CDKN1A potentiates CDK2 inhibition [42], [43], [44]. The CDKN1A gene can be transcriptionally activated by p53 [45]. ...
Background: Transforming growth factor-beta (TGFβ) is important for the morphogenesis and secretory function of the mammary gland. It is one of the main activators of the epithelial–mesenchymal transition (EMT), a process important for tissue remodeling and regeneration, but also providing cells with the plasticity to form metastases during tumor progression. Noncancerous and cancer cells respond differently to TGFβ. However, knowledge of the cellular signaling cascades triggered by TGFβ in various cell types is still limited.
Methods: MCF10A (noncancerous, originating from fibrotic breast tissue) and MCF7 (cancer, estrogen receptor-positive) breast epithelial cells were treated with TGFβ1 directly or through conditioned media from stimulated cells. Transcriptional changes (via RNA-seq) were assessed in untreated cells and after 1-6 days of treatment. Differentially expressed genes were detected with DESeq2 and the hallmark collection was selected for gene set enrichment analysis.
Results: TGFβ1 induces EMT in both the MCF10A and MCF7 cell lines but via slightly different mechanisms (signaling through SMAD3 is more active in MCF7 cells), and many EMT-related genes are expressed in MCF10A cells at baseline. Both cell lines respond to TGFβ1 by decreasing the expression of genes involved in cell proliferation: through the repression of MYC (and its targets) in MCF10A cells and the activation of p63-dependent signaling in MCF7 cells (CDKN1A and CDKN2B, which are responsible for the inhibition of cyclin-dependent kinases, are upregulated). In addition, estrogen receptor signaling is inhibited and caspase-dependent cell death is induced only in MCF7 cells. Moreover, direct incubation with TGFβ1 and treatment of cells with conditioned media similarly affected transcriptional profiles. However, TGFβ1-induced protein secretion is more pronounced in MCF10A cells; therefore, the signaling is propagated through conditioned media (bystander effect) more effectively in MCF10A cells than in MCF7 cells.
Conclusions: Estrogen receptor-positive breast cancer patients may benefit from high levels of TGFB1 expression due to the repression of estrogen receptor signaling, inhibition of proliferation, and induction of apoptosis in cancer cells. However, some TGFβ1-stimulated cells may undergo EMT, which increases the risk of metastasis.
... In normal physiological circumstances TGF-β signaling often promotes cellular quiescence and apoptosis, serving as a potent tumor suppressor [13]. TGF-β signaling can upregulate Cyclin-dependent kinases (CDK) inhibitors, such as p21 and p15, stimulating cell cycle arrest by promoting cellular quiescence [14]. At the same time, TGF-β signaling can downregulate positive cell cycle regulators, such as cyclin D1 [15]. ...
The transforming growth factor-beta (TGF-β) signaling pathway is a vital regulator of cell proliferation, differentiation, apoptosis, and extracellular matrix production. It functions through canonical SMAD-mediated processes and noncanonical pathways involving MAPK cascades, PI3K/AKT, Rho-like GTPases, and NF-κB signaling. This intricate signaling system is finely tuned by interactions between canonical and noncanonical pathways and plays key roles in both physiologic and pathologic conditions including tissue homeostasis, fibrosis, and cancer progression. TGF-β signaling is known to have paradoxical actions. Under normal physiologic conditions, TGF-β signaling promotes cell quiescence and apoptosis, acting as a tumor suppressor. In contrast, in pathological states such as inflammation and cancer, it triggers processes that facilitate cancer progression and tissue remodeling, thus promoting tumor development and fibrosis. Here, we detail the role that TGF-β plays in cancer and fibrosis and highlight the potential for future theranostics targeting this pathway.
CIP/KIP and INK4 families of Cyclin-dependent kinase inhibitors (CKIs) are well-established cell cycle regulatory proteins whose canonical function is binding to Cyclin-CDK complexes and altering their function. Initial experiments showed that these proteins negatively regulate cell cycle progression and thus are tumor suppressors in the context of molecular oncology. However, expanded research into the functions of these proteins showed that most of them have non-canonical functions, both cell cycle-dependent and independent, and can even act as tumor enhancers depending on their posttranslational modifications, subcellular localization, and cell state context. This review aims to provide an overview of canonical as well as non-canonical functions of CIP/KIP and INK4 families of CKIs, discuss the potential avenues to promote their tumor suppressor functions instead of tumor enhancing ones, and how they could be utilized to design improved treatment regimens for cancer patients.
Transforming growth factor (TGF)-β is a multifunctional cytokine expressed by almost every tissue and cell type. The signal transduction of TGF-β can stimulate diverse cellular responses and is particularly critical to embryonic development, wound healing, tissue homeostasis, and immune homeostasis in health. The dysfunction of TGF-β can play key roles in many diseases, and numerous targeted therapies have been developed to rectify its pathogenic activity. In the past decades, a large number of studies on TGF-β signaling have been carried out, covering a broad spectrum of topics in health, disease, and therapeutics. Thus, a comprehensive overview of TGF-β signaling is required for a general picture of the studies in this field. In this review, we retrace the research history of TGF-β and introduce the molecular mechanisms regarding its biosynthesis, activation, and signal transduction. We also provide deep insights into the functions of TGF-β signaling in physiological conditions as well as in pathological processes. TGF-β-targeting therapies which have brought fresh hope to the treatment of relevant diseases are highlighted. Through the summary of previous knowledge and recent updates, this review aims to provide a systematic understanding of TGF-β signaling and to attract more attention and interest to this research area.
Colorectal cancer (CRC) is a multifaceted and heterogeneous ailment that affects the colon or rectum of the digestive system. It is the second most commonly occurring form of cancer and ranks third in terms of mortality rate. The progression of CRC does not occur due to a single mutational event; rather, it is the result of the sequential and cumulative accumulation of mutations in key driver genes of signaling pathways. The most significant signaling pathways, which have oncogenic potential due to their deregulation, include Wnt/β-catenin, Notch, TGF-β, EGFR/MAPK, and PI3K/AKT pathways. Numerous drug target therapies have been developed to treat CRC using small molecule inhibitors, antibodies, or peptides. Although drug-targeted therapy is effective in most cases, the development of resistance mechanisms in CRC has raised questions about their efficacy. To overcome this issue, a novel approach of to drug repurposing has come to light, which utilizes already FDA-approved drugs to treat CRC. This approach has shown some promising experimental results, making it a crucial avenue of research in the treatment of CRC.
We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.
This chapter discusses the phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino acid composition and sequence, and (3) to determine the precise location of amino acid residues that are posttranslationally modified by fatty acid acylation, glycosylation, methylation, acetylation, or phosphorylation.
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Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.
Murine D type cyclins associate with a catalytic subunit (p34PSK-J3) with properties distinct from known cyclin-dependent kinases (cdks). Mouse p34PSK-J3 shows less than 50% amino acid identity to p34cdc2, p33cdk2, and p36cdk3, lacks a PSTAIRE motif, and does not bind to p13suc1. Cyclin D1-p34PSK-J3 complexes accumulate in macrophages during G1 and decline in S phase, whereas complexes involving cyclins D2 and D3 form in proliferating T cells. Although histone H1 kinase activity is not detected in cyclin D or PSK-J3 immunoprecipitates, cyclin D-p34PSK-J3 complexes assembled in vitro stably bind and phosphorylate the retinoblastoma gene product (pRb) and an Rb-like protein (p107) but do not interact with pRb mutants that are functionally inactive. Thus, p34PSK-J3 is a cyclin D-regulated catalytic subunit that acts as an Rb (but not H1) kinase.
The growth-suppressive function of the retinoblastoma susceptibility gene product, RB, has been implicated in the mediation
of growth inhibition and negative regulation of certain proliferation related genes by transforming growth factor-beta 1 (TGF-beta
1). Early gene responses to TGF-beta 1 were examined in order to determine their dependence on the cell cycle and on the growth-suppressive
function of RB. TGF-beta 1, which rapidly elevates the steady-state level of junB and PAI-1 mRNAs and decreases that of c-myc
mRNA, induces these responses in S-phase populations of Mv1Lu lung epithelial cells containing RB in a phosphorylated state.
Since in this state RB is presumed to lack growth-suppressive activity, the response to TGF-beta 1 was also examined in DU145
human prostate carcinoma cells whose mutant RB product lacks growth-suppressive function. In these cells, TGF-beta 1 also
decreases c-myc expression at the transcription initiation level. These results suggests that the c-myc, junB, and PAI-1 responses
to TGF-beta 1 are not restricted to the G1 phase of the cell cycle and that down-regulation of c-myc expression by TGF-beta
1 can occur through a mechanism independent from the growth-suppressive function of RB.