Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Apr;18(4):1793-801.
doi: 10.1128/MCB.18.4.1793.

Inactivation of p16 in human mammary epithelial cells by CpG island methylation

S A Foster  1 , D J Wong, M T Barrett, D A Galloway
Affiliations

Inactivation of p16 in human mammary epithelial cells by CpG island methylation

S A Foster et al. Mol Cell Biol. 1998 Apr.

Abstract

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Analysis of different E7 proteins for their abilities to allow M0 bypass in HMEC. (A) Abilities of HMEC expressing different E7 proteins to bypass M0. −, the cells behaved very similarly to uninfected or LXSN-infected controls; +, some activity, but less than that of HPV-16 E7; +++, wild-type HPV-16 E7 activity. Results are shown for uninfected HMEC6 (UN), HMEC6 infected with LXSN retroviral vector (LXSN), or LXSN containing HPV-16 E7, HPV-6 E7, HPV-16 E7D21S, HPV-16 E7Δ21–24, HPV-16 E7C24G, HPV-16 E7E26G, and HPV-16 E7H2P. (B) Western blot for cyclin A in HMEC6 expressing different E7 proteins. (C) Immunoprecipitation with HPV-16 E7 polyclonal antibody of 35S-labeled HMEC4 cells expressing the indicated E7 proteins to demonstrate expression. PRE denotes the use of nonimmunized rabbit serum. (D) Immunoprecipitation as in panel C but with HPV-6 E7 polyclonal antibody.
FIG. 2
FIG. 2
Western blots for cell cycle-related proteins in HMEC9. Cells were harvested early after establishment of the culture (passage 3) and at subsequent passages through passage 7, when the cells were predominantly in M0. The blots were probed with the indicated antibodies.
FIG. 3
FIG. 3
Expression of p16 in HMEC1 and HMEC3 before and after M0. Western blots for p16 in uninfected early-passage cells before M0 (E.P.), cells expressing E6 after escaping from M0 (E6), or cells expressing E7 or E6/E7 after bypassing M0 (E7 and E6/E7) are shown.
FIG. 4
FIG. 4
Expression of p16 in HMEC. Passage numbers are indicated above the lanes. M0 usually occurred around passage 7 to 10, as indicated; M1 for uninfected cells usually occurred around passage 20. UN, uninfected HMEC. E6 or E6/E7, HMEC infected with E6- or E6/E7-expressing retrovirus. (A) Western blots for p16 and p27. Numbers in brackets below the lanes indicate the relative amounts of p16. (B) Northern blots for p16 exon 1 with 36B4 loading control. Numbers in brackets below the lanes indicate the relative amounts of p16 mRNA normalized for 36B4. (C) p16 promoter CpG island methylation assay. U and M indicate the primer pair specific for unmethylated and methylated p16 promoter CpG islands, respectively. Molecular size markers (in base pairs) are indicated in the far left lane. H249 and H1618 are lung cancer cell lines used as controls for unmethylated and methylated p16 alleles, respectively.
FIG. 5
FIG. 5
PCR-based p16 promoter methylation analysis. (A) E7-expressing cells (HMEC1, passage 15; HMEC3, passage 23; HMEC6, passage 16; and HMEC8, passage 24). (B) Clones of E6/E7-expressing HMEC8 (passage 50). (C) E6/E7-expressing HMEC6, pooled population (passage 66), and clones thereof. Primers specific for the unmethylated (U) and methylated (M) p16 promoter regions are indicated. H249 and H1618 are described in the legend to Fig. 4.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alcorta D A, Xiong Y, Phelps D, Hannon G, Beach D, Barrett J C. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci USA. 1996;93:13742–13747. - PMC - PubMed
    1. Band V, De Caprio J A, Delmolino L, Kulesa V, Sager R. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol. 1991;65:6671–6676. - PMC - PubMed
    1. Band V, Sager R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci USA. 1989;86:1249–1253. - PMC - PubMed
    1. Banks L, Edmonds C, Vousden K H. Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells. Oncogene. 1990;5:1383–1389. - PubMed
    1. Barrett M T, Reid B J, Joslyn G. Genotypic analysis of multiple loci in somatic cells by whole genome amplification. Nucleic Acids Res. 1995;23:3488–3492. - PMC - PubMed

Publication types

Substances

LinkOut - more resources