Oncogene (2001) 20, 6164 ± 6171 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Protection against chemotherapy-induced cytotoxicity by cyclin-dependent kinase inhibitors (CKI) in CKI-responsive cells compared with CKI-unresponsive cells Mathias Schmidt1,2 and Zhen Fan*,1 1Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, TX 77030, USA Inactivation of the retinoblastoma (Rb) protein caused by Keywords: chemotherapy; chemoprotection; Rb; cell gene mutation, association with oncoproteins from small cycle; CDK DNA viruses, mutational inactivation of p16Ink4a,or overexpression of cyclin D is a common feature of many human cancer cells and is causally associated with the Introduction aberrant proliferation control of cancer cells; whereas normal cells maintain an integrated cell cycle machinery The development of neoplasia frequently involves and are subject to cell cycle checkpoint control by cyclin- inactivation of the retinoblastoma (Rb) protein and dependent kinase (CDK) inhibitors (CKIs). To determine p53 tumor-suppressor pathways and disruption of cell whether this dierence can be translated into a cycle checkpoints that monitor the integrity of therapeutic advantage to protect normal cells from replication and division of mammalian cells (Sherr, adverse cytotoxicity caused by chemotherapy, we 1994; Weinberg, 1991, 1995). These genetic alterations established cell model systems for ecdysone-inducible in neoplasia are often causally associated with expression of p16Ink4a, p21Waf1, and p27Kip1 in one CKI- uncontrolled proliferation of cancer cells. In the past responsive cell line (A431 human vulvar epidermoid decades, the design and concept of conventional cancer carcinoma cells with functional Rb) and one CKI- chemotherapy has been in principle based on defective unresponsive cell line (SiHa human cervical cancer cells control of cell cycle events in cancer cells and the with nonfunctional Rb). Expression of p16Ink4a, p21Waf1,or quantitative dierence in the proportion of fast- p27Kip1 in both SiHa and A431 cells strongly inhibited dividing cells between cancer cells and normal cells CDK2 activity, indicating functional expression of the (Darzynkiewicz, 1995; Kohn et al., 1994). While many CDK inhibitors in both cell lines. However, only in A431 chemotherapeutic drugs can successfully kill fast- cells did expression of p16Ink4a, p21Waf1, or p27Kip1 cause dividing cancer cells, the drugs unfortunately also Rb dephosphorylation, arrest cell cycle traversal, and damage several types of rapidly proliferating normal potently inhibit cell proliferation. Induction of p16Ink4a, cells such as the hematopoietic precursors, intestinal p21Waf1, or p27Kip1 in SiHa cells failed to cause Rb cells, and hair follicle cells. This nonselective killing for dephosphorylation or to arrest cell cycle traversal, and rapidly proliferating normal cells often causes serious such induction only minimally inhibited cell proliferation. adverse eects in cancer patients, such as bone marrow We then compared the chemosensitivity of clones derived suppression, anorexia (appetite loss), nausea, vomiting, from these two cell lines when the CKIs were and were diarrhea, stomatitis, and alopecia (hair loss) and thus not induced. Induction of p16Ink4a,p21Waf1, or p27Kip1 limits the doses of these drugs that can be tolerated. conferred strong resistance to paclitaxel- or cisplatin- The progression of the mammalian cell cycle is driven mediated cytotoxicity on the CKI-responsive A431 cells by periodic activation of a group of nuclear enzymes but not on the CKI-unresponsive SiHa cells. Our results called cyclin-dependent kinases (CDK) in response to support a novel chemotherapy strategy for treating various intracellular and extracellular signals (Morgan, patients with Rb pathway-impaired cancers by concur- 1995; Sherr, 1996). Cells become committed to division rent administration of chemotherapy with CKIs as at a point in mid to late G1 phase of the cell cycle (known chemoprotective agents for normal cells. Oncogene as the restriction point) following phosphorylation of the (2001) 20, 6164 ± 6171. Rb protein. The CDK inhibitors (CKIs) decelerate the cell cycle traversal by inactivating the CDKs that phosphorylate the Rb protein, and they are therefore key negative regulators of eukaryotic cell cycle traversal. *Correspondence: Z Fan, Box 36, The University of Texas MD Two families of CKIs, the INK and KIP/CIP inhibitors, Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX have been identi®ed. The INK family of CKIs includes 77030, USA; E-mail: [email protected] p16Ink4a,p15Ink4b p18Ink4c, and p14/p19Arf, which are 2Current address: Byk Gulden Lomberg Chemische Fabrik GmbH, Byk-Gulden-Str. 2, D-78467 Konstanz, Germany inactivated by gene deletion, by point mutation, or by Received 9 April 2001; revised 28 June 2001; accepted 5 July 2001 silencing through methylation in a high percentage of CDK inhibitors and chemoprotection M Schmidt and Z Fan 6165 human tumors. The KIP/CIP family consists of p21Waf1, tion of p16Ink4a,p21Waf1, or p27Kip1 in individual clones p27Kip1, and p57Kip2, which are rarely mutated in human of both cell lines with high expression levels inhibited malignancies (Sherr, 2000; Sherr and Roberts, 1999). the activities of CDK2, indicating the functional A hallmark of neoplasia is the disturbance of cell expression of these inhibitors in these cells. However, cycle controls. Although overexpression of such CKIs only in A431 cells did induction of the CDK inhibitors as p21Waf1 and p27Kip1 has been shown to cause arrest cell cycle traversal and increase drug resistance. increased resistance to chemotherapy in certain types In SiHa cells, functional expression of p16Ink4a, p21Waf1, of human cancer cells (Fueyo et al., 1998; Ruan et al., or p27Kip1 was associated with inhibited CDK activity, 1998; Schmidt et al., 2000; St Croix et al., 1996; but cell proliferation and drug sensitivity were not Waldman et al., 1997), recent studies suggested that altered. Comparing the results with those for unin- CKIs may actually be used to protect normal cells duced cells, we found that inducible expressions of from unwanted toxicity from chemotherapy and we p16Ink4a,p21Waf1, and p27Kip1 all imposed strong may thus turn this disadvantage into a therapeutic resistance to chemotherapy on the Rb-functional advantage (Davis et al., 2001; Stone et al., 1996). The A431 cells but were much less eective in the Rb- rationale for such strategy is based on the hypothesis disrupted SiHa cells. Our data provide experimental that substantial CKI-mediated chemoresistance can be support for the novel therapeutic strategy of combining found only in cells with normal Rb. In most cancer chemotherapy with inhibition of CDK as a means of cells, the Rb pathway is inactivated. (Weinberg, 1995; chemoprotection of normal cells from chemotherapy- Wiman, 1993). This disturbance is often caused by induced nonspeci®c cytotoxicity in patients with Rb- infection from small DNA viruses such as the human pathway impaired cancers. papilloma virus (HPV), by mutational inactivation of p16Ink4a or overexpression of cyclin D and, in a limited set of malignancies (such as retinoblastoma and Results osteosarcoma), by genetic mutation of the Rb gene. The inactivation of Rb allows cancer cells to evade the Establishment of p16Ink4a-, p21Waf1-, and p27Kip1-inducible normal cell cycle checkpoint control, whereas normal expression clones in A431 and SiHa cells cells have functional Rb and thus are liable to checkpoint regulation. This CKI-mediated resistance p16Ink4a, p21Waf1,orp27Kip1 cDNA was subcloned into to chemotherapy might therefore be an ideal approach the ecdysone-inducible mammalian cell expression to protect normal cells from unwanted chemotherapy- vectors, and the expression vectors were transfected induced toxicity in patients with Rb pathway-impaired in A431 and SiHa cells for stable transfectant clones. cancer. The approach may justify a novel therapeutic Over 10 clones for each cell line were obtained with strategy by concurrently treating Rb pathway-impaired high expression levels of p16Ink4a, p21Waf1, or p27Kip1 human cancer with chemotherapy in combination with upon induction. Figure 1 shows Western blot analyses inhibiting CDK activity to protect normal cells from of p16Ink4a, p21Waf1, and p27Kip1 proteins in these chemotherapy-induced cytotoxicity. representative clones (termed SiHa-p16, SiHa-p21, In this study, we employed the ecdysone-inducible and SiHa-p27, and A431-p16, A431-p21, and A431- expression system to ectopically express three repre- p27, respectively) 24 h after induction of gene expres- sentative CDK inhibitors, p16Ink4a, p21Waf1,orp27Kip1, sion with muristerone A. Induction of p16Ink4a, p21Waf1, each of which inhibits the progression of the cell cycle with a dierential timing, in selected cell lines that contain functional or nonfunctional Rb as a `proof-of- concept' experimental model. Because of technical diculties in obtaining normal human cell transfectant for inducible expression of p16Ink4a, p21Waf1, or p27Kip1 in the cells, we instead used A431 human vulvar epithelial carcinoma cells, which contain functional Rb but mutated p53 (Kwok et al., 1994). For Rb-nonfunc- tional cells, we used SiHa human cervical carcinoma cells that are known to be infected with HPV (Baker et al., 1987); the HPV virus produces two oncoproteins, E6 and