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The Candidate Tumor Suppressor Gene ZAC Is Involvedin Keratinocyte Differentiation and Its Expression IsLost in Basal Cell Carcinomas

Eugenia Basyuk,1,2 Vincent Coulon,3 Anne Le Digarcher,1 Marjorie Coisy-Quivy,2

Jean-Pierre Moles,3 Alberto Gandarillas,2,3 and Laurent Journot1

1Institut de Genomique Fonctionnelle, CNRS-UMR5203, INSERM-U661, Universite Montpellier 1,Universite Montpellier 2; 2Institut de Genetique Moleculaire, CNRS-UMR5535,Universite Montpellier 2; and 3Laboratoire de Dermatologie Moleculaire,IURC, EA3754, Montpellier, France

AbstractZAC is a zinc finger transcription factor that induces

apoptosis and cell cycle arrest in various cell lines.

The corresponding gene is maternally imprinted and

localized on chromosome 6q24-q25, a region harboring

an unidentified tumor suppressor gene for a variety

of solid neoplasms. ZAC expression is lost or

down-regulated in some breast, ovary, and pituitary

tumors and in an in vitro model of ovary epithelial

cell transformation. In the present study, we examined

ZAC expression in normal skin and found a high

expression level in basal keratinocytes and a lower,

more heterogeneous, expression in the first suprabasal

differentiating layers of epidermis. In vitro, ZAC was

up-regulated following induction of keratinocyte

differentiation. Conversely, ZAC expression triggered

keratinocyte differentiation as indicated by induction of

involucrin expression. Interestingly, we found a dramatic

loss of ZAC expression in basal cell carcinoma, a

neoplasm characterized by a relatively undifferentiated

morphology. In contrast, ZAC expression was

maintained in squamous cell carcinomas that retain

the squamous differentiated phenotype. Altogether,

these data suggest a role for ZAC at an early stage of

keratinocyte differentiation and further support its role in

carcinogenesis. (Mol Cancer Res 2005;3(9):483–92)

IntroductionZac1 is a mouse zinc finger transcription factor, which

induces apoptosis and cell cycle arrest in vitro , and abrogates

tumor formation in nude mice (1). Zac1 binds DNA and

displays transactivation or transrepression activities depending

on the precise arrangement of its binding sites (2), and

accumulating evidence suggests its involvement in the control

of cell proliferation and differentiation. Thus, inhibition of Zac1

expression by antisense oligonucleotides enhances pituitary cell

proliferation (3), and expression of Lot1, the rat orthologue of

Zac1 , is lost during the spontaneous transformation of rat ovary

surface epithelial cells in vitro (4). Interestingly, Zac1/Lot1 is

negatively regulated by epidermal growth factor, suggesting

that it might be involved in a feedback loop controlling cell

proliferation on mitogen activation (5). In addition to its role as

a transcription factor, Zac1 serves as a transcriptional

coactivator or corepressor of nuclear receptors and binds to

the nuclear receptor coactivators CBP/p300 and p160 (6). More

recently, it was suggested that Zac1 is a transcriptional

coactivator for p53 (7) and that it regulates the proapoptotic

gene Apaf-1 (8).

The human orthologue ZAC/LOT1/PLAGL1 is widely

expressed in normal tissues and the corresponding protein

binds DNA and displays transactivation activity (9). As its

mouse counterpart, human ZAC displays an antiproliferative

activity by inducing apoptosis and cell cycle arrest in

transfected cells. In addition, alternative splicing of human

ZAC transcript generates two mRNA species encoding proteins

with different number of zinc finger domains (5 versus 7),

which differentially regulate apoptosis and cell cycle arrest

(10). Noteworthy, ZAC maps to chromosome 6q24-q25 (9, 11),

a region frequently deleted in many solid tumors and thought

to harbor at least one tumor suppressor gene. Loss of

heterozygosity at 6q24-q25 occurs in breast and ovary cancer,

melanomas, astrocytomas, and renal cell carcinomas. The

functional properties of ZAC and the chromosomal location of

the corresponding gene make it a plausible candidate for the

tumor suppressor gene at 6q24-q25.

In this context, loss or down-regulation of ZAC expression is

observed in a large proportion of mammary (12) and ovary

(11, 13) tumor-derived cell lines and tumors, in nonfunctioning

pituitary adenomas (14), and in head and neck squamous cell

carcinoma (SCC; ref. 15). Expression profiling of human breast

tumors (16), lung adenocarcinomas (17), and breast tumor–

derived cell lines (16, 18) with microarrays confirmed down-

regulation of ZAC expression. We found that ZAC expression is

reinduced in breast tumor–derived cell lines by treatment with

the demethylating agent azacytidine, suggesting that the

Received 2/22/05; revised 8/5/05; accepted 8/11/05.Grant support: Centre National de la Recherche Scientifique, La LigueNationale contre le Cancer, and L’Association pour la Recherche contre leCancer (A. Gandarillas and L. Journot), La Ligue Nationale contre le Cancerfellowship (V. Coulon), and European Molecular Biology Organizationfellowship (A. Gandarillas).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Laurent Journot, Institut de Genomique Fonctionnelle,141, rue de la cardonille, F-34094 Montpellier Cedex 5, France. Phone: 33-467-142-932; Fax: 33-467-542-432. E-mail: [email protected] D 2005 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-05-0019

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methylation status of ZAC gene critically modulates its

expression (12). This hypothesis was further supported by the

demonstration of ZAC imprinting in human (19, 20) and mice

(21) and by the finding that methylation of ZAC promoter is

responsible for its observed monoallelic expression (22, 23).

Although the above-mentioned data support a role for ZAC as a

tumor suppressor gene, its precise mechanism of action and

target genes are mostly unknown at present.

Because ZAC is potentially involved in the homeostasis of

mammary and ovary epithelial cells, we assessed its

expression in different epithelial tissues, including epidermis.

Human epidermis is a stratified, constantly renewed epithelium

in which proliferation and differentiation are compartmental-

ized and tightly controlled. It consists of basal, suprabasal

(lower spinous, upper spinous, and granular), and cornified

layers. The basal layer comprises two populations of

keratinocytes: stem cells and transit amplifying cells that

initiated their differentiation program after a rapid phase of

clonal expansion (24, 25). Differentiating keratinocytes detach

from the basal membrane, increase in size, and transform into

a cornified envelope that sheds from the surface of the skin

(26). Therefore, the epidermis is an excellent model to study

the regulation of proliferation and differentiation in vivo and

its alteration during carcinogenesis.

In the present study, we analyzed ZAC expression during

differentiation of human primary keratinocytes in vitro and

in normal and pathologic skin samples, including psoriasis

and two common types of nonmelanoma skin cancer [i.e.,

basal cell carcinomas (BCC) and SCCs]. Our data suggest a

role for ZAC in keratinocyte differentiation and BCC

formation.

ResultsZAC Expression in Normal Skin

We first examined ZAC expression in five normal human

skin samples using nonradioactive in situ hybridization with

digoxigenin-labeled riboprobes. As a positive control for in situ

hybridization, we used a probe for the differentiation marker

cytokeratin K1, which prominently labeled the suprabasal

spinous layers of epidermis and, to a lesser extent, the granular

layer but not the basal layer (Fig. 1A). As a negative control,

identical samples were hybridized with a probe for lacZ that

produced no signal (Fig. 1B). In situ hybridization with the

ZAC probe showed a strong and homogenous expression in

the basal layer of epidermis (Fig. 1C and D). We detected as

well a faint and more heterogeneous expression in the spinous

layers. Interestingly, in one normal skin sample, we detected

ZAC expression in all suprabasal keratinocytes, including the

lower and upper spinous and granular layers (data not shown),

indicative of some individual variations. We also observed ZAC

expression in the dermal fibroblasts (Fig. 1D) and in the

epithelial cells of the sudoriferous glands (Fig. 1E). In contrast,

no expression was observed in the endothelium of the blood

vessels (Fig. 1F).

FIGURE 1. ZAC expression in normalskin. Sections from normal skin samples wereprocessed for in situ hybridization with probesfor cytokeratin 1 (A), h-galactosidase (B), orZAC (C-F). A-D. Epidermis. E. Sudoriferousgland. F. Blood vessel. A, B, and D-F. Thefield is 1.5 � 1.2 mm. C. The field is 3 �2.4 mm.

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ZAC Expression Is Induced in Early DifferentiatingKeratinocytes In vitro

To gain insights into the role of ZAC in epithelial cell

biology, we analyzed ZAC expression during keratinocyte

differentiation in vitro . Primary culture of human keratinocytes

is widely used to study factors that regulate stem cell

proliferation, the initiation of terminal differentiation and tissue

assembly, and the process of neoplastic transformation. Primary

keratinocytes from human foreskin were cultured under low-

calcium and low-serum conditions in a specialized keratinocyte

growth medium that keeps these cells as a monolayer, in an

undifferentiated stage, for extended periods of time (27, 28).

Terminal differentiation under these conditions can be initiated

in keratinocytes, and a proportion of cells within the monolayer

express the differentiation marker involucrin (29) and eventu-

ally shed into the culture medium (30). When the calcium

concentration is raised, cells start to form desmosomes and

stratify with selective migration of involucrin-positive cells out

of the basal layer (31). We detected low level of ZAC

expression in the keratinocyte cultures grown under low-

calcium conditions by in situ hybridization (Fig. 2B). Because

addition of calcium to the culture medium increases the

proportion of differentiating keratinocytes in the stratified

layer, this system allows the evaluation of ZAC expression

during differentiation. We did real-time reverse transcription-

PCR (RT-PCR) with total RNAs derived from control

keratinocytes cultured in low-calcium medium and from

keratinocytes at different time points after raising calcium

concentration (Fig. 2A). We observed an induction of ZAC

expression in keratinocytes stimulated with calcium as early as

8 hours following calcium addition. ZAC expression levels

remained high until 24 hours and then gradually declined at

48 and 72 hours. This finding was further corroborated by

in situ hybridization data (Fig. 2B and C). We noted a

heterogeneous distribution of ZAC signal in cells grown in the

presence of 1.5 mmol/L calcium where cells in cluster

expressed high levels of ZAC (Fig. 2C), whereas isolated

cells displayed low ZAC expression, reminiscent of the

situation in control cells (Fig. 2B).

To further assess the regulation of ZAC expression during

keratinocyte differentiation, we used another differentiation

system (i.e., anchorage-independent growth in methylcellu-

lose). In contrast to the calcium-induced stratification system,

keratinocytes grown under these conditions exhibit a certain

extent of synchronization. Keratinocytes are irreversibly

committed by 5 to 6 hours and undergo terminal differentiation

by 24 hours when most of them express the differentiation

marker involucrin (32). Total RNAs from adherent keratino-

cytes or from keratinocytes placed in suspension for different

period of time were analyzed by Northern blot hybridization

with ZAC cDNA as a probe (Fig. 3A). To control for RNA

quality and equal loading, we rehybridized the Northern blots

with a probe against 18S rRNA. We repeated the same

experiment with an independent preparation of primary

keratinocytes and analyzed ZAC expression levels using real-

time RT-PCR (Fig. 3B). In both experiments, loss of

anchorage resulted in a transient increase in ZAC expression

after 3 to 6 hours in suspension. At that time, keratinocytes

loose their ability to proliferate and become irreversibly

committed to differentiation. After 12 hours in suspension,

DNA synthesis in the keratinocytes is inhibited. At this time

point, ZAC expression decreased significantly and remained

low after 24 hours in suspension when the cells terminally

differentiate (Fig. 3A and B). Interestingly, trypsinization of

the cells rapidly up-regulated ZAC expression as evidenced by

Northern blot (Fig. 3C) or real-time RT-PCR (data not shown)

analyses.

ZAC-Induced Keratinocyte Differentiation In vitroBecause ZAC was expressed during early stages of

keratinocytes differentiation in normal skin and in primary

FIGURE 2. ZAC expression in primary keratinocytes is induced bycalcium. Normal keratinocytes were grown in vitro in a specialized mediumwith low-calcium concentration. A. ZAC expression level was quantified byreal-time RT-PCR at different time points after calcium concentration wasraised to 2 mmol/L. Normalization of ZAC expression level was done usinggeometric averaging of B2M, ARP, and hypoxanthine phosphoribosyl-transferase expression levels. Points, mean of two independent experi-ments done in triplicate; bars, SE. *, P < 0.01, Student’s t test. B and C.In situ hybridization on keratinocytes grown in low calcium (B) and24 hours after raising calcium concentration (C).

ZAC Is Involved in Keratinocyte Differentiation


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keratinocytes, we tested whether it was able to induce

keratinocytes differentiation in vitro . We transfected HaCaT,

a keratinocyte-derived cell line, with plasmids encoding ZAC

splice variants, ZAC and ZACD2 , which are both expressed in

normal epidermis (data not shown). ZACD2 is a variant of

ZAC , which lacks the two NH2-terminal zinc fingers, and

differs from ZAC in its ability to control cell cycle exit and

apoptosis (10). A green fluorescent protein–encoding vector

was transfected as a control in our experiments. Thirty-six

hours after transfection, we did immunostainings for ZAC and

the differentiation marker involucrin. The number of cells

positive for both involucrin and ZAC was determined and

compared with the number of green fluorescent protein– and

involucrin-positive cells in the control. ZAC or ZACD2expression led to a significant increase of involucrin level in

transfected cells (Fig. 4). These results suggest that ZAC

expression is sufficient to promote keratinocyte differentiation

in vitro.

ZAC Expression in Pathologic Skin SamplesTo find out whether the up-regulation of ZAC expression

observed during differentiation in vitro was associated with the

decrease of the proliferative potential and the initiation of

terminal differentiation in vivo , we analyzed ZAC expression in

hyperproliferative disorders of the skin (i.e., psoriasis and skin

tumors).

Psoriasis is a pathology of the skin characterized by an

increased proliferation rate and abnormal differentiation that

leads to thickening of the proliferative and differentiated layers

and to an increase of the epidermal turnover (33). We did in situ

hybridization on sections of psoriatic human skin biopsies. In

all five samples examined, we observed rather extended ZAC

expression with respect to normal skin. ZAC mRNA was

expressed in the thickened basal and suprabasal layers of the

psoriatic epidermis (Fig. 5A and C). The expression was strong

and homogeneous in the basal layers and much weaker and

heterogeneous in the suprabasal layers. Control hybridization

with the h-galactosidase probe produced no signal (data not

shown) and a probe for cytokeratin K1–labeled suprabasal

layers (Fig. 5B and D). Thus, we observed increased expression

of ZAC in psoriatic epidermis, consistent with the delay in the

initiation of terminal differentiation that is typical for this type

of skin pathology.

We further examined ZAC expression in two types of skin

cancer, BCCs and SCCs. Morphologically, SCC is character-

ized by an irregular and highly disorganized keratinocyte

proliferation, forming diffuse structures (34). SCC is locally

more invasive than BCC. It consists of squamous cells, which

are usually more differentiated than BCC-forming cells, and it

has a high ability to develop metastasis. We found a strong ZAC

expression in all eight SCCs examined by in situ hybridization

(Table 1; Fig. 6A and B). On one section, the epidermis

overlaying the tumor was present and we noted that ZAC was

expressed in the basal and suprabasal layers. We also observed

ZAC expression in the lymphocytes infiltrating the tumors (data

not shown). Altogether, our data suggest that ZAC is not

involved in SSC formation.

BCC is the most common type of human cancer (35).

Morphologically, this tumor is characterized by tumor nodules

FIGURE 3. ZAC expression is regulated by anchorage-independentgrowth. A and B. Normal keratinocytes were grown in a specializedmedium containing methylcellulose for the indicated period of time. A.Total RNAs were prepared and analyzed by Northern blotting with a ZACprobe and a 18S rRNA probe. B. ZAC expression level was quantified asin Fig. 2A but for the control genes B2M, ARP, glyceraldehyde-3-phosphate dehydrogenase, and PP1A. Points, mean of a representativeexperiment done in triplicate; bars, SE. *, P < 0.01, Student’s t test. C.Northern blot analysis of ZAC and 18S rRNA expression in keratinocytesbefore and after trypsin treatment.

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inf iltrating dermis, with characteristic peripheral palisading.

BCC nodules are composed of small nondifferentiated cells

with a basal cell-like phenotype (36). Familial BCCs, called

Gorlin syndrome or nevoid BCC syndrome, have loss-of-

function mutations of PATCHED (PTCH), which encodes a

SONIC HEDGEHOG (SHH) receptor (37, 38). We examined

13 sporadic and 5 familial BCCs by in situ hybridization and

found loss of ZAC expression in all sporadic BCC (Table 1).

ZAC expression was absent from all tumor nodules but weakly

present in the basal and suprabasal layers of the epidermis

overlaying the tumor on the same tissue section (Fig. 6C-E). In

several cases, ZAC expression was not detected even in the

epidermis overlaying the tumor. In the biopsies of BCC taken

from patients with Gorlin syndrome, ZAC expression was

completely lost in three of five cases. In the other two cases,

it was strongly diminished when compared with normal skin

taken from the same patients where expression was noted in the

basal and suprabasal layers of epidermis (Fig. 6F). In these

cases, a very weak ZAC expression was observed in the middle

of the tumoral nodules but not on their periphery where most

proliferating cells appear to be located (39).

ZAC Expression Is Not Regulated by the SHH PathwayConstitutive activation of the SHH pathway is sufficient to

induce BCC in transgenic mice (40) and human skin (41). The

observed loss or decrease of ZAC expression in all BCC

examined suggested a possible down-regulation of ZAC by the

activation of the SHH pathway. To test this hypothesis, we

analyzed ZAC expression in primary keratinocytes grown in the

presence of recombinant SHH. It was shown previously that

SHH opposes calcium-induced cell cycle arrest in differentiat-

ing keratinocytes (42). Primary keratinocytes were grown to

60% to 80% confluence in low-calcium conditions before the

calcium concentration was raised to 1.5 mmol/L and SHH was

simultaneously added to the culture medium. Total RNAs from

cells cultivated with or without SHH for 4 and 24 hours were

analyzed by RT-PCR. To test for RNA integrity, each RNA

sample was analyzed for actin expression. No effect on ZAC

expression was observed after addition of SHH to the culture

medium (Fig. 7). To verify that recombinant SHH was active in

this experiment, the up-regulation of BMP-2B , a member of the

transforming growth factor-h family and a known target of

SHH in mammals (43), was verified and confirmed by RT-PCR

(Fig. 7).

DiscussionWe undertook the present study to test a possible

involvement of ZAC in epidermal differentiation and to

evaluate its implication in the development of skin cancer.

We detected ZAC expression in the basal and, to a lower level,

suprabasal layers of normal epidermis. Using two models of

keratinocyte differentiation in vitro , we observed an early up-

regulation of ZAC expression following induction of differen-

tiation. Interestingly, in both models, ZAC induction is transient

and followed by a down-regulation. These observations are in

good agreement with the distribution of ZAC mRNA in normal

skin. Keratinocytes initiate differentiation within the basal layer

where ZAC is abundantly expressed. The differentiation process

FIGURE 4. ZAC promotes involucrin expression in HaCaT cells.HaCaT cells were transfected with green fluorescent protein (GFP ),ZAC, or ZACD2 and stained with 4V,6-diamidino-2-phenylindole (blue ),anti-ZAC (red), and anti-involucrin (green) antisera. Top, high-magnifi-cation pictures of ZAC- and ZACD2-positive cells; bottom, involucrin-positive cells and transfected cells (i.e., green fluorescent protein – orZAC-positive cells) were scored and the ratios of involucrin-positivecells to transfected cells were calculated for each condition. Columns,mean of three independent experiments; bars, SD. *, P < 0.01, Student’st test.



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proceeds during the migration of these cells toward the outmost

layers of the skin where ZAC expression gradually disappears.

In addition, we showed that ZAC expression in an immortalized

keratinocyte cell line promotes keratinocyte differentiation.

Collectively, this set of data suggests that ZAC expression

contributes to keratinocyte differentiation.

We found a complete loss or a significant decrease in ZAC

expression in all sporadic and familial BCCs examined. This

observation is in agreement with the proposed tumor

suppressor function for ZAC and with the observed loss or

down-regulation of ZAC expression in breast, ovary, and

pituitary tumors (12–14). BCC is the most common type of

neoplasm and its incidence is dramatically increasing. BCC

metastasis occurs only locally and sporadically in relation to

sun exposure. Its incidence is increased significantly in some

rare genetic disorders, such as Gorlin syndrome. Familial

BCCs and about one-third of sporadic BCCs have loss-of-

function mutations of PATCHED (37, 38), which encodes a

receptor for the morphogen SHH. Activation of PATCHED

negatively regulates the activity of SMOOTHENED, a seven-

transmembrane domain receptor controlling the activation of

the GLI family of transcription factors (44, 45). Moreover,

human sporadic BCCs consistently express GLI-1 (46). In

agreement with these data, overexpression of Shh (40) or GLI-1

(47) is sufficient to induce BCC-like tumors in transgenic mice.

Interestingly, ZAC and GLI-1 display opposite patterns of

expression. Indeed, GLI-1 expression is not detected in basal

keratinocytes of the normal skin, which display high expression

of ZAC in our in situ experiments. Conversely, GLI-1 is

expressed in all sporadic and familial BCCs, where ZAC is lost.

We thus assessed whether the SHH-PTCH-SMOH-GLI path-

way modulates ZAC expression and found no regulation of

ZAC by recombinant SHH. Hence, ZAC loss of expression is

apparently not directly controlled by the aberrant activation of

the SHH pathway in BCC.

Besides PTCH , very few other tumor suppressor genes have

been involved in BCC formation and/or development. One

interesting candidate is TP53 for which a specific, UV-induced

mutation spectrum has been reported in sporadic BCC (48, 49).

The recent demonstration that mouse Zac1 behaves as a p53

coactivator (7) suggests that the loss of ZAC expression in

sporadic BCC may correlate with a decrease in the p53-induced

response to DNA damage. Noteworthy, Zac1 cotransfection

was reported to potentiate p53-mediated stimulation of

p21WAF1/Cip1 expression (7), which is involved in keratinocyte

differentiation (50).

Our data regarding ZAC expression in vitro and in vivo

suggest a role for ZAC in the early stages of keratinocyte

differentiation. Due to its functional properties (1, 9, 10), one

would predict that ZAC would be involved in the cell cycle

arrest, which usually accompanies the induction of terminal

differentiation in most tissues. Indeed, in normal epidermis, the

progressive loss of ZAC expression in the suprabasal layers

could be correlated with an irreversible proliferation shutdown.

However, several lines of evidence indicate that ZAC is most

likely not directly involved in negative regulation of cell cycle

progression in the epidermis. First, the maintaining of ZAC

expression in the hyperproliferative psoriasis and SCC indicates

FIGURE 5. ZAC expression in psoriasis sam-ples. Two psoriasis samples (A-D) were analyzedby in situ hybridization for ZAC (A and C) andcytokeratin 1 (B and D) expression. A-C. Thefield is 1.2 � 1.2 mm. D. The field is 2.4 � 2.4 mm.

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no link between ZAC expression and a proliferation block.

Second, epidermal differentiation is not invariably accompanied

by a complete withdrawal from the cell cycle, because

keratinocytes retain the ability to synthesize DNA (51, 52)

and continue to grow in size during differentiation (26). Finally,

SCCs and psoriasis retain the capacity to differentiate and

express ZAC , whereas BCC cells display a nondifferentiated

phenotype and do not express ZAC . All these data suggest that

ZAC plays a role in early events of keratinocyte differentiation,

at a step distinct from the control of the cell cycle progression.

Interestingly, keratinocytes of suprabasal layers of normal and

psoriatic epidermis and SCC display increased cell size,

whereas BCCs are formed by small-sized cells. ZAC may thus

have a role in the cellular growth that takes place at the

initiation of differentiation that is altered in SCCs and lost in

BCCs.

In addition, it was shown recently that cytokeratin 14 is a

direct target of mouse Zac1 and that several other cytokeratins

are positively or negatively regulated by Zac1 (2). Consistent

with the putative role of its human counterpart in keratinocytes

differentiation, Zac1 was observed to repress cytokeratin 19,

which has been proposed as an interfollicular stem cell marker

(53), and to activate cytokeratin 14, a component of the basal

layer where ZAC is also present (24).

Finally, our data also unveil a potential role for the loss of

the candidate tumor suppressor gene ZAC in the development

of BCC. Additional experiments aiming at identifying ZAC

target genes will provide insights into the process of

keratinocyte differentiation that is lost in BCC.

Materials and MethodsCell Culture

HaCaT keratinocyte cells were maintained in DMEM plus

10% fetal bovine serum and transfected using Lipofectamine

2000 (IVG, Cergy, France). Plasmids encoding ZAC and

ZACD2 were described previously (10). Monolayer cultures

of primary human keratinocytes were prepared from neonatal

foreskins as described (54). Cells were maintained in serum-

free keratinocyte growth medium K-SFM (IVG). Stratifica-

tion was induced by addition of 2 mmol/L calcium or 10%

fetal bovine serum to the medium. Cells were harvested for

RNA isolation at different time points after induction. To

study the effect of SHH on ZAC expression, the keratino-

cytes were grown to 60% to 80% confluence before the

concentration of calcium in the medium was raised to 1.5 to

2 mmol/L. Recombinant SHH (Research Diagnostics, Inc.,

Flanders, NJ) was simultaneously added at a final concen-

tration of 20 Ag/mL. Cells were harvested for RNA isolation

at different time intervals after SHH addition. To induce

terminal differentiation, the keratinocytes were trypsinized

and maintained in suspension in 1.75% methylcellulose as

described previously (32).

RNA Isolation and Northern Blot AnalysisTotal RNA was isolated from keratinocyte cultures using

Trizol (LTI, Cergy, France). Northern blots of total RNAs (20

Ag) were hybridized with a human ZAC probe (nucleotides 684-

2,800 of the human cDNA; Genbank accession no. AJ006354)

and a probe for 18S rRNA. Hybridization was in Church buffer

[0.5 mol/L phosphate buffer (pH 7), 7% SDS, 2% bovine serum

albumin] and final washing step was at 65jC in 1� SSC and

0.2% SDS.

Real-time and Semiquantitative PCR AnalysiscDNAs were generated from 1 Ag total RNAs treated with

DNase I by using random hexamers and Moloney murine

leukemia virus reverse transcriptase (LTI). Real-time PCR was

done using Applied Biosystems (Courtaboeuf, France) SYBR

Green PCR mix according to the manufacturer’s instructions.

Housekeeping genes used to normalize ZAC expression levels

were selected using the geNorm process (55). The sequences of

the primers for real-time PCR are ZAC 5V-CCACTCACAG-GAGCTGATGAAA and 5V-GCAGCCTTCAGTTGGAAT-

GAA, B2M 5V-TGACTTTGTCACAGCCCAAGATA and 5V-CGGCATCTTCAAACCTCCA, ARP 5V-CTCCAAGCAGATG-CAGCAGA and 5V-CCCATCAGCACCACAGCC, hypoxan-

thine phosphoribosyltransferase 5V-CGGCTCCGTTATGGCGand 5V-GGTCATAACCTGGTTCATCATCAC, and PP1A 5V-GTCGACGGCGAGCCC, 5V-TCTTTGGGACCTTGTCTGCAA,and glyceraldehyde-3-phosphate dehydrogenase 5V-TGTTC-GACAGTCAGCCGC and 5V-GGTGTCTGAGCGATGTGGC.The sequences of the primers for semiquantitative PCR are

Table 1. ZAC expression in normal and pathologic skinsamples was assessed by in situ hybridization and visualscoring

n Diagnosis ZAC expression

308 BCC �281 BCC �280 BCC �221 BCC �220 BCC �392 BCC �314 BCC �394 BCC �466 BCC �366-2 BCC �436 BCC �218bs BCC �448 BCC �354 BCC (NBCCS) �355 BCC (NBCCS) �357 BCC (NBCCS) +/�315 BCC (NBCCS) �318 BCC (NBCCS) +/�366-1 SCC +430 SCC +++432 SCC ++345 SCC +397 SCC +287 SCC +442 SCC ++431 SCC +467 Normal skin ++442n Normal skin +455 Normal skin ++428 Normal skin ++356 Normal skin (NBCCS) +++408 Normal skin (scalp) +427 Normal skin (scalp) +

NOTE: �, no ZAC expression; +/�, very weak ZAC expression; +, ++, and +++,increasing levels of ZAC expression.Abbreviation: NBCCS, a sample from a patient with nevoid BCC (Gorlin)syndrome.



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ZAC 5V-AACCGGAAAGACCACCTGAAAAACCAC and

5V-GTCGCACATCCTTCCGGGTGTAGAA (amplicon 303 bp),

actin 5V-ACTGGCATCGTGATGGACTCC and 5V-GTTG-GCGTACAGGTCTTTGCG (amplicon 444 bp), and BMP-2B

5V-CACCATGATTCCTGGTAACC and 5V-TCTCCAGATG-TTCTTCGTGG (amplicon 390 bp). Semiquantitative PCR

was done as follows: 2 minutes at 94jC followed by 20 to

30 cycles: 30 seconds at 94jC, 30 seconds at 55jC for BMP-2B

primers, 57jC for ZAC primers, and 60jC for actin primers, and

30 seconds at 72jC. Lower numbers of cycles were used to verify

linearity of the amplification process. Half of the PCR reaction

was run on a 6% polyacrylamide gel and analyzed by Southern

blotting.

In situ HybridizationIn situ hybridization on sections of normal skin, psoriasis,

and skin tumors obtained after biopsy was done as described

previously (56). For detection of ZAC , we used a 294-

nucleotide– long RNA probe, corresponding to nucleotides

1,889 to 2,182 of the human cDNA (Genbank accession no.

AJ006354). The specificity of this probe was shown

previously (56). For detection of cytokeratin 1, we used a

FIGURE 6. ZAC expression in skin tumors. TwoSCCs (A and B), two sporadic BCCs (C and D), aBCC from a patient with Gorlin syndrome (E), andnormal skin from the same patient (F) were analyzedfor ZAC expression by in situ hybridization. Sectionswere counterstained with hematoxylin. Arrows, tumornodules in BCC samples; arrowheads, tumor on SCCsections.

FIGURE 7. ZAC expression is not repressed by recombinant SHH.Normal keratinocytes were grown in low-calcium medium. The calciumconcentration was raised to 1.5 mmol/L for the indicated period of time inthe presence (+) or absence (�) of recombinant SHH. Total RNAs wereisolated and analyzed by RT-PCR for ZAC, actin, and BMP-2B expression.

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1-kb probe corresponding to the coding region of cytokeratin

1 cDNA, which was hydrolyzed before hybridization.

Following ISH, sections of BCC and SCC were stained

with hematoxylin.

ImmunocytochemistryCells grown on coverslips were fixed with 4% formaldehyde

in PBS for 30 minutes, washed with PBS thrice, and

permeabilized with 0.1% Triton in PBS for 10 minutes. Double

labeling was done with anti-ZAC rabbit polyclonal antibodies

(diluted 1:1,000) and anti-involucrin mouse monoclonal anti-

bodies SY5 (Interchim, Montlucon, France), diluted 1:100 in

PBS containing 1% bovine serum albumin. Secondary anti-

bodies anti-rabbit FITC (Sigma, San Quentin Fallavier, France)

and anti-mouse CY3 (Sigma) were diluted 1:300 and 1:3,000,

respectively, in PBS with 1% bovine serum albumin. No cross-

reactivity was observed in control experiments. The coverslips

were mounted in 4V,6-diamidino-2-phenylindole–containing

medium (56).

AcknowledgmentsWe thank Drs. Edouard Bertrand, Jean-Marie Blanchard, and Annie Varrault forcritical reading of the article and helpful comments.

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2005;3:483-492. Mol Cancer Res Eugenia Basyuk, Vincent Coulon, Anne Le Digarcher, et al. Basal Cell CarcinomasKeratinocyte Differentiation and Its Expression Is Lost in

Is Involved inZACThe Candidate Tumor Suppressor Gene

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