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Molecular Biology and Genetics |
Department of Pathology and University of California at San Francisco Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94143-0511 [C. R. H., K. C., T. D. T.]; Ohio State University Medical Center, Columbus, Ohio 43210 [G. J. N.]; and The Johns Hopkins Comprehensive Cancer Center, Baltimore, Maryland 21231 [M. E., S. B. B., J. G. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subsequent to the proliferation arrest, vHMECs proliferate an additional 30–50 generations beyond the time that the bulk population activates the proliferative arrest. These cells eventually reach a second population growth plateau that we previously termed agonescence (2) and that is phenotypically different from human mammary fibroblast senescence and the HMEC first plateau. Agonescent vHMEC populations have both high proliferation and high death indexes, although they exhibit no net increase in cell number (1) . Furthermore, nearly 100% of vHMECs approaching agonescence exhibit chromosomal defects, including aneuploidy, telomeric associations, and various other classes of structural abnormalities (1) . Such chromosomal instability is reminiscent of the abundant and heterogeneous chromosomal changes observed in premalignant and malignant breast cancer lesions (8 , 9) .
To gain insight into the origin of vHMECs, we asked whether this cell subpopulation exists before the first population growth plateau. We found that this vHMEC subpopulation exists before this proliferation barrier. Seeking to extend our in vitro observations to the tissue from which HMECs are cultured, we examined histologically normal mammary tissue for p16 promoter hypermethylation, a defining characteristic of vHMECs. We report here that a significant fraction of normal women contain mammary epithelial cells with p16 hypermethylation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fluctuation Analysis.
Fluctuation analysis experiments (Fig. 1, A and B
, SET 2) were conducted by (a) imposing a population bottleneck on early-passage HMEC populations; (b) allowing in vitro expansion of the initial populations, subcultivating the cell populations as needed to prevent confluence; until (c) the cultures ceased increasing in cell number (1)
. In parallel with SET 2 of samples 184 and 48, mass cultures were propagated (Fig. 1, Aand B
, SET 1) under standard culture conditions. Although in vitro propagation of cells, by its very nature, favors cells with a higher proliferation rate, we postulate here that selection for vHMECs occurs predominantly at the first growth plateau. We make this postulation because the proliferation rates and colony-forming efficiencies of early-passage HMEC populations and early-passage vHMEC populations are equivalent (data not shown). When the cell populations ceased expansion, colonies were fixed and stained by standard protocols. Colonies were scored positively if they met the following criteria: colony diameter 
6 mm; staining significantly darker than background; and microscopic confirmation that >90% of the cells in the colony were uniformly small. To validate these colony-scoring criteria, we performed immunocytochemical staining for p16 on representative colonies. Only those colonies that met these criteria were p16-negative (data not shown).
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Binomial distributions were calculated according to the formula (using one Poisson assumption, because x << n:
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Immunohistochemical Analysis.
p16 immunostaining was performed on 5-µm sections of paraffin-embedded tissue using the p16INK4a Ab-4 antibody (clone 16P04; NeoMarkers, Inc., Fremont, CA). Briefly, deparaffinized slides were blocked with 3% hydrogen peroxide, followed by heat-mediated antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0). Slides were incubated for 1 h at room temperature with a 1:200 dilution of the antibody in PBS and 1% BSA. Antibody staining was visualized using biotinylated-antimouse antibodies (Vector Laboratories, Burlingame, CA) and ABC-HRP Elite (Vector Laboratories, Burlingame, CA), followed by diaminobenzidine reaction. Sections were counterstained with light hematoxylin and then dehydrated and coverslipped with mounting medium.
MSP.
The p16 CpG island methylation status was assessed by using a modification of the protocol described previously (10)
. Briefly, DNA was extracted according to standard protocols, denatured by NaOH, modified by sodium bisulfite, purified using Wizard DNA purification resin (Promega), treated again with NaOH, precipitated with ethanol, and resuspended in water. A nested approach was used, first amplifying the bisulfite-modified DNA with the flanking primers 5'-AGA AAG AGG AGG GGT TGG TTG G-3' (upper primer) and 5'-ACR CCC RCA CCT CCT CTA CC-3' (lower primer), "R" being a mixture of A and G. After this step, 4 µl of each 1:1000-diluted flanking PCR reaction was used as a template for MSP, using the primers previously described (10)
. Ten µl of each PCR reaction were loaded onto nondenaturing 6% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination.
MSP-ISH.
Formalin-fixed, paraffin-embedded tissues (5-µm sections) were used to determine the incidence and cellular distribution of p16 hypermethylation in clinical samples. MSP-ISH was performed as described previously (11)
. After pepsin digestion of specimens, the DNA was bisulfite modified (CpG Wiz p16 methylation assay; Intergen Discovery Products, Gaithersburg, MD). After a manual hot start (94°C, 3 min), 35 cycles were conducted (55°C, 1.5 min; 94°C, 1 min). PCR used the methylation-specific primer set described previously (10)
. After PCR, ISH was performed using a methylated allele-specific internally digoxigenin-labeled probe (1 µg/ml), diluted with Hybrisol VII (Ventana Medical Systems). The amplicon and probe were codenatured (95°C, 5 min), hybridized (37°C, 2 h), washed (1x SSC +2% BSA, 52°C, 10 min), incubated with antidigoxigenin alkaline phosphatase conjugate (1:200; Roche Molecular Biochemicals), and then exposed to the chromogen, nitroblue tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Enzo Diagnostics) at 37°C. The final counterstain, nuclear Fast Red, stains the negative cells pink in contrast to the blue signal. To confirm normal histology, adjacent serial sections were stained with H&E, as per standard histological procedures. To confirm staining specificity, adjacent serial sections were treated as above, but omitting the PCR amplification step.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Luria-Delbrück fluctuation analysis is a combined experimental and analytical approach that can be used to determine the origin of variant cells that are resistant to a selective pressure (12
, 13)
. Cells are grown under two sets of conditions, SET 1 and SET 2 (Fig. 1A)
, and then are analyzed for their ability to generate variants. In SET 1 growth conditions, the cells are aliquoted from a mass culture immediately before the selection pressure, thereby measuring the frequency of resistant cells. These aliquots represent a random sampling of cells from the mass population at that time, and the number of variants per aliquot should display a binomial distribution (where, under the condition of a low frequency event, the variance, V, approximates the mean, m).
In SET 2 growth conditions, cells are aliquoted into many replicates of small subpopulations of founder cells (population bottlenecks) and are allowed to proliferate for several generations before the selection pressure. If the variant cells are generated by conditions found at the arrest (adaptation), then previous propagation history should be irrelevant and each subpopulation would have an equal probability of generating variants. The adaptation model predicts that the number of colonies per subpopulation in SET 2 will exhibit a binomial distribution (V 
m). In contrast, under the selection model, if the variant cells preexist before the selective pressure, then the variability from subpopulation to subpopulation in SET 2 will be higher than that predicted by the binomial distribution (V > m).
To apply fluctuation analysis to the question of the origin of vHMECs, we defined the selective pressure as the self-imposed proliferative arrest of the first population plateau, thereby operationally making the first period of exponential proliferation the nonselective period (Fig. 1B)
. We then measured the frequency and variance of the vHMECs that grew beyond the proliferative arrest. HMECs were cultured according to SET 2 and SET 1 conditions as follows: replicate subpopulations of small numbers of HMECs (1.0 x 103 - 1.3 x104; Table 1
; SET 2) were cultured separately, while the parental population was cultured in parallel en mass, (SET 1). Cell populations were allowed to proliferate exponentially (subcultivated as necessary), until the cell number ceased to increase and the cells became large and vacuolated (i.e., they activated the proliferative arrest; data not shown). Cultures were then fed regularly until colonies of p16-negative vHMECs (data not shown) were clearly distinguishable from the background (14–21 days). At this time, the plates were fixed, stained, and scored for the frequency of cells that could grow beyond the proliferation barrier (Fig. 1C)
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m; Table 1
), the distribution of colonies among the SET 2 subpopulations did not (V>m; Table 1
; P < 0.001 by 
2 test for each experiment). Similar observations were made in nine independent experiments, four of which are summarized fully here (Table 1)
. These data are consistent with the model that vHMECs are not generated at the time of the proliferative arrest but, rather, exist within the population before the arrest and are selected at the arrest. For this reason, we will henceforth in this paper use the terms "preselection" and "postselection" to describe HMECs in vitro, in keeping with their original description (3)
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Histologically Normal Human Mammary Tissue Contains Epithelial Cells with Hypermethylated p16 Promoters.
The magnitude of the colony formation frequencies observed in the bottleneck populations of the fluctuation analysis suggested that variant cells not only existed in the population before the proliferative arrest but also were present very early within the culture, and perhaps even in vivo. To test the hypothesis that these variant cells exist in vivo, we assessed mammary tissue from reduction mammoplasty patients (a patient population with no overt increased risk for breast cancer; Ref. 14
) for a defining characteristic of postselection HMECs, namely p16 promoter hypermethylation (4, 5, 6, 7)
. We used the sensitive MSP assay (10)
to ascertain p16 promoter methylation status in DNA isolated from histologically normal mammary tissue sections. Strikingly, we detected methylated p16 promoter sequences in DNA isolated from 7 of 15 women (47%; Fig. 2A
; Table 2
). All of the samples that contained methylated-specific PCR product also contained unmethylated-specific PCR product, indicating a mixture of methylated and unmethylated alleles (Fig. 2A
and data not shown).
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; Table 2
). Seventy-one % (10 of 14) of reduction mammoplasty specimens analyzed by MSP-ISH contained undetectable levels of methylation (see Fig. 2I
; Table 2
). Adjacent serial sections were stained with H&E to allow cytological evaluation of the regions of positive staining. Microscopic analysis revealed that methylated p16 alleles were present in histologically normal mammary epithelial cells in both lobular (Fig. 2, E–G)
and ductal (data not shown) regions. Neither myoepithelial cells nor stromal cells (including fibroblasts) contained detectable methylated alleles (Fig. 2G)
. Although several of the breast reduction samples were positive by both the MSP and MSP-ISH analyses, five samples demonstrated discordance between the two methods of analysis (Table 2)
. Possible explanations for this discordance include differential sensitivities of the two techniques, the apparent focal nature of the hypermethylated cells, and/or sampling differences.
We then analyzed the distribution pattern and approximate frequency of cells positively staining for p16 promoter methylation within the tissue and exhibited the data using a tissue map (Fig. 3)
. This method of presenting the data allows a display of spatial information and heterogeneity. In general, the samples fell into three major categories: (a) as already mentioned, the majority of samples (10 of 14) contained an undetectable number of cells per histological section with methylated p16 promoter sequences (<<1% positive epithelial cells per section; e.g., Figs. 2, H and I
, and 3, A–G
, and data not shown). By calculating the total area occupied by epithelial cells per section and the mean number of epithelial cell nuclei per unit area, we estimate that the average histological section contains 
30,000 epithelial cell nuclei. Thus, in 10 of the 14 samples, the frequency of detection is less than 1/30,000, or 3.3 x 10-5. It is currently unknown whether repeated sampling from different sites of the same breast will reveal similar or different frequencies; (b) two samples contained rare foci or an intermittent scattering of cells with methylated p16 promoter sequences (samples 9624 and 5308; Fig. 3H
and data not shown; note frequent juxtaposition of methylated clusters and unmethylated clusters); and (c) finally, two samples contained a considerable number of cells per section (10–50% positive epithelial cells per section, although the frequency varied greatly from region to region; samples 9698 and 10811; Fig. 3, I and J
). Large adjoining regions of positivity (e.g., as outlined by the green dashed line in Fig. 3I
) may indicate clonal origin of variant cells or a field effect.
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, sample 10811; and data not shown). Specimens with undetectable hypermethylation varied in their levels of p16 protein expression; some contained low-to-undetectable expression levels (data not shown), whereas others expressed p16 abundantly in focal patches (e.g., Fig. 3K
, sample 10966). The basis for the induction of p16 expression in mammary tissue is currently under investigation.
To confirm the specificity of the MSP-ISH results, an adjacent serial section from each reduction mammoplasty was processed in parallel, but omitting the PCR step. Omission of PCR resulted in the loss of nuclear hybridization (Fig. 2, K and M)
, thereby attesting to the specificity of the post-PCR methodology. Furthermore, with each set of MSP-ISH reactions, we processed a cervical CIS sample, shown previously by various methods to contain extensive p16 promoter hypermethylation, as well as a benign cervical sample previously shown to contain unmethylated promoter sequences (11)
. As expected, after MSP-ISH, the cervical CIS sample showed abundant nuclear hybridization (Fig. 2O)
and the signal was undetectable in the benign cervical tissue (Fig. 2N)
. In further control experiments, the CIS sample stained negatively when (a) PCR was omitted, (b) primers were omitted from the PCR, or (c) the sodium bisulfite modification reaction was omitted (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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If the p16 promoter-hypermethylated variant epithelial cells indeed represent precursors to breast cancer, our observations suggest that premalignant breast lesions are more frequent than generally appreciated. Studies by Nielsen et al. (22) and Alpers and Wellings (23) have shown a surprising degree of undetected premalignant and malignant lesions. In the Nielsen study of double mastectomy specimens from 110 medicolegal autopsies, in which the cause of death was unrelated to breast cancer, nearly one-third of the women harbored hyperplastic lesions (32%), more than one-quarter contained atypical ductal hyperplasia (27%), almost one-fifth showed ductal CIS (18%), and 2% had overt invasive breast cancer. Furthermore, almost one-half of the women with ductal CIS had bilateral (41%) and/or multifocal (45%) disease (22) . Alpers and Wellings’ study of 185 breast samples from random autopsies confirmed this high prevalence of undetected premalignant breast lesions (23) . Other studies have reported lower frequencies of premalignant lesions (24 , 25) , but sampling methods and clinical definitions varied among these studies (26) . These data indicate that the initiation of premalignant lesions, identified by morphological alterations within the tissue, is by no means a rare event.
Our observation of p16-methylated variant cells in histologically normal tissue may be identifying premalignant lesions before the morphological changes reported above. Several recent studies have shed light on the genomic status of histologically normal breast tissue. Deng et al. (27) showed that a common genomic alteration in primary invasive breast cancers (loss of 3p) often occurred in adjacent morphologically normal ductal tissue. Using a broader range of markers, Larsen et al. (28) showed that 22% of microdissected histologically normal breast samples showed microsatellite instability and/or loss of heterozygosity. Kandel et al. (29) , furthermore, showed that p53 mutations, including missense mutations previously detected in breast cancer, could be detected in normal and benign breast tissue. These observations, along with the epigenetic alteration reported here, support the hypotheses that early premalignant breast lesions are more frequent, and harbor more genetic and epigenetic alterations, than previously suspected. We anticipate that further study of vHMECs in vitro and in vivo will continue to provide insights into early changes in breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Avon Foundation, The Cancer League, Inc., the California Cancer Research Program, and Grant CA73952 from NIH/National Cancer Institute awarded to T. D. T. G. N. was supported by the Lewis Foundation. C. R. H. was supported by a Howard Hughes Medical Institute Pre-Doctoral Fellowship. 
2 Present address: Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center (CNIO), 28029 Madrid, Spain.
3 To whom requests for reprints should be addressed, at Department of Pathology, University of California at San Francisco, 513 Parnassus Avenue, Box 0511, San Francisco, CA 94143-0511. Phone: (415) 502-6115; Fax (415) 502-6163; E-mail: ttlsty{at}itsa.ucsf.edu
4 The abbreviations used are: HMEC, human mammary epithelial cell; vHMEC, variant HMEC; p16, p16INK4a (also known as CDKN2A and MTS-1); MSP, methylation-specific PCR; ISH, in situ hybridization; CIS, carcinoma in situ; PD, population doubling.
Received 10/18/02. Accepted 1/27/03.
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 V. M. Weaver and P. Gilbert Watch thy neighbor: cancer is a communal affair J. Cell Sci., March 15, 2004; 117(8): 1287 - 1290. [Abstract] [Full Text] [PDF]
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 A. K. Meeker, J. L. Hicks, E. Gabrielson, W. M. Strauss, A. M. De Marzo, and P. Argani Telomere Shortening Occurs in Subsets of Normal Breast Epithelium as well as in Situ and Invasive Carcinoma Am. J. Pathol., March 1, 2004; 164(3): 925 - 935. [Abstract] [Full Text] [PDF]
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