Differential Expression and Cell Cycle Regulation of

[CANCER RESEARCH54, 5816-5820, November 15, 1994)

Advances in Brief

Differential Expression and Cell Cycle Regulation of the Cydlin-dependent Kinase 4 Inhibitor p161nk4 Sun W@Tam,'

Jerry W. Shay,2 and Michele

Pagano―@

Mitotiz Inc., Cambridge, Massachusetts 02139 (S. W. T., M. P.), and the Department of Cell Biology and Neurosciences, The University of Texas Southwestern Medical Center at

Dallas, Dallas, Texas 75235 (J. W. S.)

Abstract

p2l@' (11), humanCdk2 (12), and humanCdk4 (13) and the monocbonal antibody against human cydlin Dl, clone DCS-6 (14), have been described

p16― (inhibItor of cydlin-dependentkinase 4) Is a celi cycle regulator

that specificallybinds to and inhibits Cdk4. Recently, the human mtsi

previously.

Cell Culture and SynchrOniZatiOn. Normal human foreskin melanocytes, normalhumanbreastductalepitheial cells, normalhumanbronchialepitheial

(multiple tumor suppressor 1) gene, deleted or mutated in various pri mary tumors and in a large number of transformed cell lines, was found to be Identical to ink4. In this study we have surveyed by immunoblotting the protein levels of p16@4 in normal and transformed human celia. We

cells, normal human epidermal keratinocytes, normal human umbilical vein

determined

that p16'@4 was differentially

growth

rived

different

from

tissues,

expressed

in contrast

to another

in diploid cells de cell cycle

endothelialcells, normalhumanforeskinfibroblasts,andnormalhumanaortic smooth muscle cells were obtained from Clonetics Corp. and cultured in the medium

recommended

by the manufacturer.

All cell types were cal

inhibitor,

turedfor four passagesor less (passagedat a 1:4 ratio)before proteinextrac p2l@', which Is ubiquitously expressed. In some tumor cell lines p16@h1k4tion. Normalhumanosteocytes and normalhumanT lymphocyteswere gifts protein was not detected, presumably because ofa homozygous deletion of of F. Della-Ragioneand M. Giunta. Its gene By contrast, it was found to be overexpressedin other cell lines Humanbreastepithelialcell line HBL-100; humanbreastadenocarcinoma when compared to levels in their normal counterparts. Interestingly, high cell line MCF-7;humanbreast-infiltratingductalcarcinomacell line BT549; levels of p16―― protein correlated with functional inactivation of the humanbreastcarcinomacell lines MDA-MB-134,MDA-MB-453,andMDA retinoblastoma

gene product.

We also found that p16I@@k4 protein expres

MB-468; human osteogenic sarcoma cell lines SAOS-2 and U-2-OS; human

sian varies during the cell cyde pealdng during S phase. These results show a functional relationship between p16@4 and the retinoblastoma gene product

and Indicate

that p16―4 is required

for Cdk4 inhibition

fibrosarcoma cell line HS-913-T; human leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1B;humanepithelioidcervicalcarcinomacell line HeLa;human cervical

only at the G1-S transition at the time when Cdk4 kinase activity is no longer necessary.

epidermoid

carcinoma

cell lines CaSki

and C-4-ll;

and human

em

bryonic kidney adenovirustype 5-transformedcell line 293 were obtained fromthe AmericanType CultureCollectionandculturedin Dulbecco's mod ified Eagle's medium supplemented with 10 to 20% F@S, 2 mM glutamine, 100

Introduction The gene encoding p161―4,the previously identified inhibitor of human cyclin-dependent kinase 4 (1), has recently been mapped to 9p2l, the site for the multiple tumor suppressor (Mts) locus (2, 3). The ink4 gene has been found deleted or mutated in large percentage of cell lines including melanomas, lymphomas, and breast, bladder, and kidney carcinomas (2, 3). Further investigations have shown that, although with a lower incidence, the p16I@@@c4 gene is also deleted or mutated in uncultured esophageal squamous cell carcinomas (4), lung carcinomas (5), and bladder cancers (6). Cyclins and their catalytic partner proteins, Cdks,@regulate the cell cycle in eukaryotic cells. Progression through G@requires the activity of cyclin D1-Cdk4 kinase (reviewed in Ref. 7). In contrast to p2l―@ which is able to inhibit in vitro all cyclin/Cdk complexes (8—10), p16―4 selectively inhibits in vitro Cdk4 kinase activity (1). In this report, we present our study on the differential expression and cell cycle regulation of p16@4 protein in normal and tumor human cells.

Materials and Methods Immunoreagents. The rabbit polyclonal antisera against human pl6@'4 (1) were purchasedfrom Pharmingen,Inc. The polyclonalantiseraagainsthuman

units/mbpenicillin, and 1 @.tg/ml streptomycin. Human lung fibroblasts (IMR-90) were obtained from the American Type Culture Collection at PDL 20 and grown for not more than 40 total PDLS.Cells were countedat each passage and the increasein PDL was calculatedas the bog2-foldincreasein cell number.IMR-90cells were infectedwith DNA viral proteinsand grown as describedpreviously(15—17). IMR-90 cells were synchronized in G0-G1 by incubating for 3 days in Dulbecco's modified Eagle's medium containing 0.2% FCS and stimulated to re-enter the cell cycle by addition of 10% FCS. Extract Preparation and Immunoblotting. Cell extracts were prepared as described (18). Briefly, 3 to 5 volumes of lysis buffer [50 m@iTris-HC1 (pH 7.4)-0.25 M NaC1-0.1% Triton X-100-1 mM EDTA-50 mM NaF-1 mM dithio

threitol-0.1 mMNa3VO4]were added to a cell pellet. The following protease inhibitorswere added: 0.1 mM phenylmethylsulfonylfluoride; 1 g.@g/ml of leupeptin; 10 @&g/mi of soybean trypsin inhibitor; 10 @&g/mi of L-1 chlor-3-(4-

tosylamido)-4-phenyl-2-butanone; 10 gig/miof L-1 chior-3-(4-tosylamido)-7amino-2-heptanone

hydrochloride;

1 pg/mi

of aprotinin.

After incubation

on

ice for 30 mm, the samples were centrifugedat 14,000 rpmin an Eppcndorf microfuge for 5 min at 4°Cto recover the supernatant. Proteins were trans ferred from gel to a polyvinyl difluoridemembrane(Du Pont) by semidry blotting as described in Ref. 19. Filters were subjectedto immunobbotting using the enhancedchemiluminescence(Amersham)detectionsystem accord ing to the manufacturer'sinstructions.Gels were scanned and bands were quantifiedby using a pdi laser densitometer. Results and Discussion

Received 9/2/94; accepted 10/5/94. The costs of publicationof this articlewere defrayedin partby the paymentof page charges.This articlemust thereforebe herebymarkedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by the Human Science Frontier Program Grant RO-496/93. 2 Supported

in part

by

the

NIH

Grant

CA50195

and

the

Susan

0.

Komen

Breast

Cancer

Foundation. 3 To whom requests for reprints should be addressed. 4 The

abbreviations

used

are:

Cdk,

cyclin-dependent

PDL, population doubling; BrdUrd, 5-bwmodeoxyuridine.

kinase;

F@S,

fetal

calf

serum;

Differential tissue expression of p161@@k4 has not been characterized. To this end, we have examined by immunobbotting the protein levels of p16@4 in nine different primary cultures, representative of three major tissue types: epithelial (foreskin melanocytes, breast ductal epithelial cells, bronchial epitheial cells, and epidennal keratino cytes); connective (umbilical vein endothelial cells, osteocytes, T lymphocytes, and foreskin or lung fibroblasts); and muscular (aortic 5816

Downloaded from cancerres.aacrjournals.org on March 11, 2015. © 1994 American Association for Cancer Research.

CELL CYCLE REGUlATION OF @16@@k4

pl&―@4 even after very long exposure, confirming that lack of pl&@@k4 is a common feature of transformed cell lines (2, 3). SAOS-2 cells showed low levels, whereas BT-549, MDA-MB-468, and HS-913-T showed significantly higher levels of pl61'@4protein when compared to their normal counterparts. Interestingly, these three latter cell lines have a nonfunctional retinoblastoma gene product (pRb) and p53 (see p16'@4proteinlevel was muchlower thanin othercell types.T Table 1). To confirm that inactivation of tumor suppressor genes was corre lymphocytes did not show any detectable p161nk4even after very long lated with high levels of p16I@k4protein, we analyzed p161nk4levels in exposure. Interestingly, of all normal diploid human cells examined thus far, only T lymphocytes express Cdk6 kinase (17, 20) which is other cell lines known to have perturbed pRb and p53 functions. HBL-100, a cell line expressing the SV4O large T antigen, HeLa, and the closest homologue of Cdk4 and seems to be the major catalytic C-4-II expressing the HPV-18 E6 and E7 proteins, CaSki expressing partner of D-type cyclins in these cells. Thus, it is conceivable that T the HPV-16 E6 and E7 proteins, and 293 cell line expressing the lymphocytes do not express p16@4 but instead express a homologue that is not detectable with the antiserum used in this study. p2l―@, adenovirus E1A and E1B proteins were chosen because E7 and E1A viral proteins are known to inactivate pRb, E6 and ElS viral proteins another cdk kinase inhibitor (8—10,21—24),did not show significant differential expression among the nine cell types. inactivate p53, and large T antigen inactivates both pRB and p53 (for To examine whether transformed human cell lines exhibit pl61―@4 a review see Ref. 25). In all five cell lines high levels of p16I@@@c4 protein levels similar to those of their normal counterparts, we se were detected (Fig. 1B). A summary of these results is presented in Table 1. lected ten transformed cell lines of epithelial (five breast carcinomas), connectival (two osteosarcomas and one fibrosarcoma), and smooth Finally, we examined levels of pl&―@4 protein in human lung diploid fibroblasts (IMR-90) infected with defective retroviruses cx muscular origin (two leiomyosarcomas) (Fig. 1B). By immunoblot ting, six of ten cell lines (MCF-7, MDA-MB-134, MDA-MB-453, pressing the HPV-16 E6 or E7 proteins, either alone or in combina tion, or with a control retrovirus, as previously reported (16, 17) (Fig. U-2-OS, SKLMS-1, and SKUT-1-B) showed undetectable levels of

smooth muscle cells). To obtain statistically significant results, pri mary cultures were isolated at least twice from different donors. fluorescence activated cell sorter analysis showed that at the time of harvesting all cell types were actively growing with similar percent ages ofcells in the G1, S. and G2-M phases of the cell cycle (see Table 1). As shown in Fig. lÀ, in breast and bronchial epithelial cells

Table 1 p'6 andcyclin Dl protein abundancc in primary cultures and tumor celi linesLevel ofLevel01502-MpRbp53p16of

DlCell typeOrigin(%)(%)(%)function―function―proteinproteinNormalMelanocytesEpithelial (neural crest)761212+“++++++Breast

(ectoderm)691021++±++Bronchial ductalEpithelial cellsEpithelial (ectoderm)522226+++++KeratinocytesEpitheial (ectoderm)631819++++±Endotheial cellsConnective (mesoderm)571429++++++T

lymphocytesConnective622216++——(mesoderm)OsteocytesConnective691912+++++++(mesoderm)FibroblastsConnective

(mesoderm)652114+++++++Smooth (mesoderm)571924+++++++TransformedHBL-100Breast muscleMuscular

linec611920——+++++—McF-7Breast epithelial Carcinoma511534++—++BT-549Breast

Carcinoma511732—-+++++—MDA-MB-134Breast Carcinoma75817+?—++++MDA-MB-453BreastCarcinoma621127+——++MDA-MB-468Breast

Carcinoma522127——++++±SAOS-2Osteosarcoma512524——±—U-2-OSOsteosarcoma552124++-+HS-913-TFibrosarcoma582022--+++±SKLMS-1Leiomyosarcoma

HeLaLeiomyosarcoma Cervical Carcinoma'@61 Carcinomae591427——++++±C-4-llCervical Carcinoma―561232-—+++++±293Kidney epithelial ±CaShCervical

5616

29+

1523

——

——

+++++++

Lin/541531——++++±Tra@IMR-90

vectorConnective582616++÷++++IMR-90+E6Connective611524+-+++++++IMR-90+E7Connective661123—++++++±IMR-90÷E6+E7Connective562222——+++++±IMR-90 +

+ LTConnective513118--+++++± @

a

@

b

for the state of pRB and p53 functions of a very

strong

signal;

++,

detection

had been reported previously of a strong

signal;

by Tam et al., 1994.

+, detection

of a weak

signal;

±, detection

of a very

weak

signal;

—,no signal

detected;

++++

signal much stronger than in the normal counterpart. The relative intensity of the immunoblotting signals was the average of several experiments. C SV4O

@

large

T

antigen-positive

cells.

E6- and E7-positive cells. a HPV-16

E6-

and

E7-positive

cells.

1E1A and E1B adenovirus-positive cells.

5817

Downloaded from cancerres.aacrjournals.org on March 11, 2015. © 1994 American Association for Cancer Research.

or more,

CELL CYCLE REGULATION OF @16@1@k4

A

@, @)

4_#ø

&@

@a)

4.J

@-

@8@E

‘a,

@

0 @c'.c @c'a (@W@WO&..a) a)0@-V-@4-' @.

a)

‘I,

a)L@ £..

u

— 0 £@

p16

p21

B @

0

@

-@

5-

@a@iz3€@ IL