(A) Repression of CD44 promoter activity with p53 in the human osteosarcoma cell line SAOS-2 lacking endogenous p53 expression. Human CD44 promoter (CD44P, 0- 2021 bp upstream of translation initiation codon) was fused to a firefly luciferase gene in pGL3 vector. The CD44P construct was co-transfected with CMV-vector-based constructs bearing either normal or specific-DNA-binding-defective p53 genes into the SAOS-2 cells. The cells were lysed 30h after transfection and analyzed for luciferase activity (mean±SD, n=4).
(B) The consensus sequence of p53-mediated transcriptional regulation (upper sequence), a potential non-canonical p53-binding site in the CD44 promoter, 239–263bp upstream from the first transcription initiation site (lower sequence), and the introduced point mutations (in red, lower sequence) used to inactivate the potential p53-binding site.
(C) Promoter reporter assay with normal CD44 promoter or mutated CD44P-M1 constructs in SAOS-2 cells co-transfected with or without the p53 expression vector (mean±SD, n=4). The basal activity of promotorless construct was subtracted from the presented values of CD44 promoter activity.
(D) Gel-shift analysis of p53 protein interaction with CD44P-derived, ³²P-labeled oligonucleotides with normal or mutated p53-binding site.
(E) Competition analysis of p53 protein interaction between labeled CD44P-derived oligonucleotide and non-labeled oligonucleotides bearing either normal or mutated p53-binding site sequence.

(A) Western blotting analysis of CD44 and p53 in cytokine-starved cells cultured in the presence or absence or EGF for 4h.
(B) Cell growth (mean±SD. n=3) of BPEC-T cells stably infected with lentiviral constructs expressing shRNA against CD44 or p53 were analyzed after 72h incubation in the presence or absence of EGF.
(C–D) Apoptosis (mean±SD, n=3) of BPEC-T cells exposed to doxorubicin at in the absence of 1.2 ng/ml EGF, analyzed by TUNEL assay after 24h.

A549 cells were infected with a lentiviral vector expressing an shRNA to firefly luciferase (shLuc), to p53 (shp53) or to CD44 (shCD44-2 and shCD44-3). The infection efficiency was more than 95% for all constructs (data not shown).
(A) The level of p53 and CD44 expressions in individual infected cell populations in vitro was analyzed by western blotting 72h after the infection.
(B) Individual lentivirus-infected cells (10⁶ cells per injection) were injected in nude mice and tumor weights (mean±SEM) were analyzed 4 weeks after the injection. Tumor incidence per injection is indicated in parentheses.

(A) Immunofluorescent detection of CD44 and nuclei (DAPI) in BPEC-T cells stably infected with the control shRNA against firefly luciferase (shLuc), p53 shRNA (shP53), E6 oncoprotein- or GFP-expressing constructs in subconfluent cultures.
(B) Immunofluorescent analysis of CD44 and p53 protein co-expression in BPEC-T cells.
(C–D) Western blotting analysis of total CD44 protein (C) and semi-quantitative RT-PCR analysis of total CD44 mRNA expression (D) in confluent BPEC-T cells with normal and inhibited p53 expression. The cDNAs for RT-PCR were PCR amplified from 3.3 and 1.1ng of respective cDNA.
(E) Immunohistochemical analysis of CD44 expression in mammary fat pads. The epithelial and non-epithelial compartments of mammary glands from p53−/− or p53+/+ Balb/c mice were immunostained for the epitope present in all CD44 known isoforms by the IM7 antibody and counterstained with hematoxylin.

(A) BPEC-T cells stably infected with retroviral vectors expressing either the standard form of CD44 (CD44s), control GFP, or lentiviral vectors expressing an shRNA directed against p53 (shp53) or control shRNA (shLuc) were analyzed for expression levels of p53 and CD44 by western blotting. The CD44 with higher Mw than CD44s is either a product of differential post-translational modification of CD44s or an alternatively spliced variant form of endogenous CD44, whose expression is up-regulated by ectopic expression of CD44s.
(B) Cell growth (mean±SD, n=3) of BPEC-T cells variants in the presence or absence of EGF analyzed after 72h.
(C and D) Apoptosis (mean±SD, n=3) of cells exposed to doxorubicin in the presence EGF (0.6ng/ml), analyzed by TUNEL assay after 24h.

BPLER cells were infected with a lentiviral vector expressing an shRNA to firefly luciferase (shLuc) or to CD44 (shCD44-2 and shCD44-3). The infection efficiency was more than 95% for all constructs (data not shown).
(A) The level of CD44 expression in individual infected cell populations in vitro was analyzed by western blotting 72h after the infection.
(B) Individual lentivirus-infected cells (10⁶ cells per injection) were injected in nude mice and tumor weights (mean±SEM) were analyzed 4 weeks after the injection. Tumor incidence per injection is indicated in parentheses.
(C) Hematoxylin and eosin staining of the resulting tumors. Scale = 500μm.
(D) The level of CD44 expression in tumors arising from individual infected cell populations was analyzed by western blotting 72h after the infection.
(E) Tumor-initiating frequency of individual infected cell populations.
(F, G) Soft agar colony assay of individual lentivirus-infected cells. The cells (10⁵/6cm dish) were plated in culture medium in soft agar and cultured for 4 weeks. The assay was terminated when colonies of control cells reached 1mm in diameter, at which point they were counted (mean±SEM, n+3).

(A) The p63 protein expression was inhibited by two independent shRNAs in pLKO1-Puro lentiviral vector in BPEC-T cells. The cells were lysed and analyzed for p63 and CD44 protein expression by western blotting one week after infection with viral shRNA constructs.
(B) The scheme of the function of p53-CD44-p53 axis in immortalized BPEC-T cells. Unknown signals, dependent on a cell’s position within an epithelial cell cluster, trigger induction of p53 expression, which leads, in turn, to CD44 repression, slower proliferation and increased apoptosis in response to a strong genotoxic stress. Conversely, increased CD44 expression can inhibit p53 stability by stimulating Her2-dependent activation of MDM2 protein expression (Mayo and Donner, 2001; Zhou et al., 2001).
(C) The summary of CD44 function in tumor cells. In highly tumorigenic BPLER cells with SV40 LTg-inactivated p53, CD44 is essential for anchorage-independent growth, tumor growth kinetics as well as tumor-initiating ability. In A549 cells, suppression of p53 expression accelerates tumor growth, which is dependent on elevated CD44 expression resulting from its derepression occuring in the absence of p53. The expression of CD44 is positively regulated by p63 in BPEC-T cells, in BPLER cells and in A549 cells, but the detailed molecular mechanism of this regulation in not known.