See
“An Overview” for more information on the
derivation of these cell types.
Live
cells for distribution are sent frozen (in dry ice) in ampoules containing 5 x
10e5 or 1 x 10e6 cells. Under special circumstances, we can arrange to send
growing cells in flasks. For some cell types, RNA is also available upon
specific request.
See
"REVIEW
section VIII." for more information on cell shipments.
1. Pre-stasis (Chart
1, Panel C)
Reduction
mammoplasty derived HMEC grown in a serum-containing medium, M87A or M85 (Stampfer
et al. 1980; Stampfer 1982; Garbe et al. 2009)
We
have large batches of pre-stasis HMEC to distribute at passages 4-8; most
growth ceases around passages 13-16.
Lower passage amps are usually sent to allow growth of stocks in the
recipient’s lab. Pre-stasis HMEC contain a range of phenotypes, particularly at
lower passages. Cells with luminal
marker make up ~10-25% of the population.
They are genomically stable. I
most commonly distribute cells from specimens 184, 48R, and 240L (ages
16-21). More limited quantities of HMEC
derived from older woman are also available.
2. Post-stasis: Extended Life (EL) (p16 silenced/mutated)
(Chart 1, Panel A)
Reduction
mammoplasty derived HMEC from specimen 184 that were exposed to the chemical
carcinogen benzo(a)pyrene in primary culture.
(Stampfer
& Bartley 1985, 1988; Brenner et al., 1998; Novak et al. 2009)
Very
limited quantities of the EL cultures are available. These include: 184Aa (p16 mutated), the
precursor of 184A1 and other 184A- lines; 184Be (p16 silenced), the precursor of 184B5 and other 184B- lines,
and 184Ce, the precursor of the184CeMY1 line. Talk with me directly about these
cells. More 184Aa is available than any
other EL type. These cells were exposed to BaP and so likely harbor unknown
errors.
3. Post-stasis: Post-selection (p16 silenced) (Chart 1, Panel B)
Reduction
mammoplasty, non-tumor mastectomy, or benign tumor derived HMEC, grown in a
serum-free medium, that emerge from populations at stasis following silencing
of the p16 promoter.
(Hammond et al. 1984; Romanov et al. 2001; Garbe et al. 2007, 2009;
Li et al. 2007)
We
have large batches of post-selection HMEC frozen at around passages 7-10 that
we distribute. Depending upon the individual, these cells cease active growth
around passages 14-25 (about 3 PD per passage).
These cells are routinely available from women of different ages. I most commonly distribute cells from
specimens 184, 48R, 239, 240L and 161.
These
cells are not normal. They have overcome
the stasis barrier and show many differences from the normal pre-stasis HMEC
(e.g., gene expression and promoter methylation). They express a predominantly basal phenotype,
although some luminal marker may also be present at higher passages (e.g.,
mucins and keratin 18). They become
genomically unstable as they approach the telomere dysfunction senescence
barrier. Post-selection HMEC have also
been referred to as vHMEC. These are the
cell type sold commercially as “normal primaries” although they are neither
normal nor primaries.
4. Post-stasis: GSE22 (p53 expression inhibited by GSE22)
(Chart 1, Panel A)
Pre-stasis
HMEC grown in MM that were transduced with GSE22 and show clonal escape from stasis.
In
2 separate experiments pre-stasis 184 HMEC were transduced with a genetic
suppressor element (GSE22) that produces a peptide that interferes with p53
function.. In both cases there were a
few clonal outgrowths when the vast majority of the cells ceased growth at
stasis. These cells have not been
well-characterized. They express low but
detectable p16. These cells are not routinely distributed.
5. Post-stasis: p16sh (p16 expression inhibited by shRNA
to p16) (Chart 1, Panel C)
Pre-stasis
HMEC grown in a serum-containing medium that were exposed to shRNA to p16 and
show uniform bypass of stasis.
In
2 separate experiments pre-stasis 184 HMEC were transduced with shRNA to p16
(once in M85 medium, batch 184F; once in M87A+X medium, batch 184D). These cells have not been
well-characterized. They express
increased telomerase (TRAP) activity compared to the parental pre-stasis HMEC.
These cells are not routinely distributed but can be made available.
B.
FINITE LIFESPAN HUMAN MAMMARY FIBROBLAST CELLS
(HMFC)
We
have available for distribution stocks of fibroblast cells from several
reduction mammoplasty specimens for which HMEC are available, e.g., specimens
184, 48, 240, and 161. These cells are grown in a serum-containing medium. In theory, fibroblast stocks can be obtained
from any of our specimens, including the mastectomy derived tissues, but we
have not grown up stocks to distribute from more than a few. Frozen cells are available around passages
5-8, and they senesce around passages 12-20 (2-3 PD per passage) depending upon
the individual. These cells grow slower
than the HMEC, and we do not generally have as large stocks available.
We
have very limited quantities of
frozen organoids, and are therefore very reluctant to distribute any of this
material, but will make exceptions for specific studies. More primary tissue is
available from reduction mammoplasties than mastectomies. Talk with me directly
about these cells.
D.
IMMORTALLY TRANSFORMED CELL LINES
(Stampfer & Bartley 1985, 1988; Walen & Stampfer 1989;
Lehman et al. 1993; Brenner et al. 1998; Stampfer et al. 1997, 2001, 2003;
Garbe et al. 1999, 2007; Nonet et al. 2001; Olsen et al. 2002; Chin et al.
2004; Li et al. 2007; Novak et al. 2009)
1.
Immortal lines derived from cells grown in MM, following exposure to oncogenic
agents (Chart 1, Panel A)
1a)
Lines derived from cells exposed to benzo(a)pyrene (BaP); i.e. from the
post-stasis EL cultures
184A1 (Stampfer & Bartley
1985)
Unlimited
quantities of later passages of this immortal cell line are available for
distribution. Very limited quantities of
early passage newly immortal (pre-conversion) cells are available upon specific
request. Clonal isolates are also
available. These cells are wild type for
p53 and RB, and are not anchorage independent or tumorigenic. They have the most stable karyotype of our
BaP-exposed immortally transformed lines.
This line is derived from the EL precursor 184Aa (p16 mutated).
184A1-RF (Olsen et al. 2002)
184A1was transduced with an activated Raf-1 construct under the control of inducible ER. These cells now have AIG and reduced EGF requirements for growth.
184A1-GSE22 (Stampfer et al. 2003)
184A1was
transduced with GSE22. When the GSE22
is introduced pre-conversion (passage 12) conversion proceeds rapidly. When the
GSE22 is introduced into fully immortal 184A it can provide a matched p53(-)
culture to the p53(+) 184A1. These cells are not routinely distributed but can
be made available.
184A1-hTERT(12p) (Stampfer et al. 2001; Olsen et al 2002)
Early
passage pre-conversion (passage 12) 184A1 was transduced with hTERT, resulting
in immediate uniform immortalization without undergoing conversion. They remain
sensitive to OIS. These cells are not routinely distributed.
184A1-hTERT(22p) (Stampfer et al. 2001)
184A1
already in the process of conversion (passage 22) was transduced with
hTERT. These cells proceeded through
conversion to become fully immortal. These cells are not routinely distributed.
184A1- E6; -E7; -T; -E1A (Garbe et
al. 1999)
Early passage pre-conversion (passage 12) 184A1 was transduced with either the HPV16 -E6, -E7, SV40T, or E1A genes. These viral oncogenes produce numerous effects that may accelerate or alter the conversion process. These cells are not routinely distributed.
184AA4 (Stampfer et al. 2003)
Unlimited
quantities of later passages of this immortal line are available for
distribution. Early passage cells are
not available. These cells are wild type for p53, and RB, and are not anchorage
independent or tumorigenic. They have
many karyotypic abnormalities. This
line is derived from the EL precursor 184Aa.
184AA2 (Stampfer et al. 2003)
Unlimited
quantities of this p53(-/-) immortal line are available for distribution. These
cells are wild type for RB, and have AIG.
The karyotype is unstable. 184AA2
is derived from the same EL precursor population (184Aa) as 184A1 and 184AA4.
184Aa was transduced with retroviral vectors that gave insertional mutagenesis
at the p53 locus. We presume this line acquired addition errors at crisis.
184AA3 (Stampfer et al. 2003)
Unlimited
quantities of this p53(-/-) immortal line are available for distribution. These cells are wild type for RB, and have
AIG by passage 50. The karyotype is
unstable. 184AA3 is derived from the
same EL precursor population (184Aa) as 184A1 and 184AA4. 184Aa was transduced
with retroviral vectors that gave insertional mutagenesis at the p53 locus. We
presume this line acquired addition errors at crisis.
184AA5-7
These
are immortal lines that arose in finite lifespan post-stasis EL 184Aa
populations transduced with control retroviruses. They have not been characterized, but their
early rapid growth suggests they are p53(-) like 184AA2 and 184AA3, and are a
consequence of insertional mutagenesis. These cells are not routinely
distributed.
184AaGS1-3
Finite
lifespan post-stasis EL 184Aa was transduced with GSE22 and produced three
immortally transformed cultures.
Immortalization is clonal, and each isolate is likely to be somewhat
different. We presume these lines have
acquired addition errors at crisis. These lines have not been
well-characterized and are not routinely distributed.
184AaMY1-5 (Garbe et al. in prep)
Finite
lifespan post-stasis EL184Aa was transduced with c-myc on multiple occasions;
each time there has been efficient relatively rapid immortalization of the
population. These lines have not been
well-characterized. They are not routinely distributed but can be made
available.
184AaZN1-3 (Nonet et al. 2001)
Finite
lifespan post-stasis EL184Aa was transduced with the breast cancer -associated
oncogene ZNF217 on multiple occasions. Immortalization is clonal, and each
isolate is likely to be somewhat different.
We presume these lines have acquired additional errors during the period
of agonescence. The lines examined are p53(+). They are not routinely
distributed but can be made available.
184AaE6 (Garbe et al. 1999)
Transduction of the HPV16 E6 gene into post-stasis EL 184Aa produces common immortalization following crisis. These cells are not routinely distributed but can be made available.
184B5 (Stampfer & Bartley
1985)
Unlimited
quantities of later passages of this immortal line are available for
distribution. More limited quantities of
early passage newly immortal (pre- and mid- conversion) cells are available
upon specific request. Clonal isolates
are also available. These cells are wild type for p53, and RB, and are not
anchorage independent or tumorigenic.
They have a low level of karyotypic instability. This line is derived
from the EL precursor 184Be.
184B5-erbB2
184B5
was transfected with the breast cancer associated oncogene erbB2; these cells
exhibit AIG.
184B5ME was isolated from anchorage
independent colonies of 184B5-erbB2 grown in methocel. They have not been well-characterized. Unlimited quantities are available.
184BE1 (Garbe et al. in prep)
This
is a new line of indefinite lifespan that emerged from EL 184Be. It is not yet well-characterized; it’s
gradual conversion process is consistent with a p53(+) status. It is not yet
available for distribution.
184BEMY1 (Garbe et al. in prep)
Finite
lifespan EL184Be was transduced with c-myc, producing efficient relatively
rapid immortalization of the population.
This line has not been well-characterized and is not yet available for
distribution.
184CEMY1 (Garbe et al. in prep)
Finite
lifespan EL184Ce was transduced with c-myc , producing efficient relatively rapid immortalization of
the population. This line has not been well-characterized and is not yet
available for distribution. We have not derived any “spontaneous” lines from
184Ce (culture of 184Ce has been more limited than 184Aa or 184Be).
1b)
Line derived from cells exposed to GSE22
184FGS1 (Garbe et al. 2007)
1c)
Line derived from cells exposed to hTERT
184FTERT (Stampfer et al. 2001)
Pre-stasis 184F grown in MM
were transduced with hTERT at passage 3. A clonal population maintained growth
after the
vast majority of the cells ceased growth at stasis. The population
gradually lost expression of p16 while gaining resistance to TGFß growth
inhibition. These cells have not been
well-characterized. This line is not routinely distributed but can be
made available.
2.
Immortal lines derived from post-selection HMEC grown in serum-free MCDB170 (Chart
1, Panel B)
2a)
Lines derived from post-selection HMEC transduced with c-myc and/or ZNF217
184MY1
184ZN4-7 (Nonet et al. 2001; Chin et
al. 2004)
Finite
lifespan post-selection 184 was transduced with ZNF217 on multiple
occasions. In 6 independent experiments 4 clonal immortal lines
emerged;
each isolate is likely to be somewhat different. We presume these lines acquired additional
errors during the period of agonescence. The lines examined are p53(+).These lines are not routinely distributed but can be
made available.
Finite
lifespan post-selection 184 was transduced with ZNF217, followed by c-myc, on
multiple occasions. In 5 independent experiments 4 clonal immortal lines
emerged. Three of these lines emerged
soon after the exposure to c-myc, prior to agonescence; Southern analysis
indicated they are clonal. The 2 lines
examined by array CGH show no alterations in copy numbers. We presume no additional genomic mutations
were required for immortalization. These
lines are
not routinely distributed but can be made available on a collaborative basis
upon request.
184ZNMY3-N
We
have transduced 184ZNMY2 and 184ANMY3 with oncogenic erbB2 (neu). These lines then acquired AIG. CGH analysis
shows no copy number alterations.
2b)
Lines derived from post-selection HMEC transduced with hTERT
184BTERT; 48RTERT; 161HTERT
Transduction
of hTERT into post-selection p16(-) HMEC produces efficient
immortalization bypassing the genomic instability of agonescence,
and conversion. The cells remain
sensitive to OIS. Unlimited quantities of 184BTERT are available for
distribution.
2c)
Line derived from post-selection HMEC transduced with HPV-E6
184-E6
Transduction of the HPV16 E6 gene into post-selection p16(-) HMEC produces common immortalization following crisis, bypassing conversion. These cells are not routinely distributed but can be made available.
3.
Immortal lines derived from cells grown in the less stressful serum-containing
M85/M87/M87A media ± oxytocin (X) (Chart 1, Panel C)
These lines are not yet available for routine
distribution but can be made available on a collaborative basis upon request.
3a)
Lines derived from cells following
transduction with c-myc and/or shRNA to p16 (Garbe et al. in prep)
184FMY2
Pre-stasis
184F growing in M85 were transduced with c-myc.
There was clonal escape from stasis, yielding an immortal line.
184Fp16s1
Pre-stasis
184F growing in M85 were transduced with shRNA to p16, producing post-stasis
populations. In one instance, a clonal
immortal line emerged around the period of agonescence.
184Fp16sMY
Pre-stasis 184F growing in M85 were transduced with shRNA to p16, then c-myc, quickly producing increased telomerase activity and uniform immortalization, with no CGH changes.
3b)
Lines derived from cells following transduction with hTERT (Garbe et al. in prep)
184DTERT
Pre-stasis
184D growing in M87A+X were transduced with hTERT.
Selected
Publications:
Stampfer, M.R., Hallowes, R. and Hackett, A.J., Growth of Normal Human Mammary Epithelial Cells in Culture. In Vitro 16:415-425, 1980. PMID: 6993343
Stampfer, M.R., Cholera Toxin Stimulation of Human Mammary Epithelial Cells in Culture. In Vitro 18:531-537, 1982. PMID: 6288550
Hammond, S.L., Ham, R.G., and Stampfer, M.R., Serum-free Growth of Human Mammary Epithelial Cells: Rapid Clonal Growth in Defined Medium and Extended Serial Passage with Pituitary Extract. Proc. Natl. Acad. Sci. (USA) 81:5435-5439, 1984. PMID: 6591199
Stampfer, M.R., and Bartley, J.C., Induction of Transformation and Continuous Cell Lines from Normal Human Mammary Epithelial Cells after Exposure to Benzo(a)pyrene. Proc. Natl. Acad. Sci. (USA) 82:2394-2398, 1985. PMID: 3857588
Stampfer, M.R., and Bartley, J.C., Human Mammary Epithelial Cells in Culture: Differentiation and Transformation. Cancer Treat Res 40: 1-24, 1988. PMID: 2908646
Walen, K.H., and Stampfer, M.R., Chromosome Analyses of Human Mammary Epithelial Cells (HMEC) at Stages of Chemically-induced Transformation Progression to Immortality, Cancer Genet. Cytogen. 37:249-261, 1989. PMID: 2702624
Lehman T, Modali R, Boukamp P, Stanek, J., Bennett, W.P., Welsh, J.A., Metcalf, R.A., Stampfer, M.R., Fusenig, N., Rogan, E.M., Reddel, R., and Harris, C.C. p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14, 833-839, 1993. PMID: 8504475
Stampfer, MR, Bodnar, A, Garbe, J, Wong, M, Pan, A, Villeponteau, B, Yaswen, P, Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells, Mol Biol Cell 8:2391-2405, 1997. PMID: 9398663
Brenner, AJ, Stampfer, MR, Aldaz, M, Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with inactivation, Oncogene 17:199-205, 1998. PMID: 9674704
Garbe, J, Wong, M, Wigington,
D, Yaswen, P, Stampfer, MR, Viral oncogenes accelerate conversion to
immortality of cultured conditionally immortal human mammary epithelial cells,
Oncogene 18:2169-2180, 1999. PMID: 10327063
Romanov, SR, Kozakiewicz, K, Holst, CR, Stampfer, MR,
Haupt, LM, Tlsty, TD, Normal human mammary epithelial cells spontaneously
escape senescence and acquire genomic changes, Nature 409:633-637, 2001. PMID:
11214324
Nonet, GH, Stampfer, M, Chin, K,
Gray, JW, Collins, CC, Yaswen, P, The ZNF217 Gene amplified in breast cancers
promotes immortalization of human mammary epithelial cells, Cancer Res. 61:
1250-1254, 2001. PMID: 11245413
Stampfer, MR, Garbe, J, Levine, G,
Lichtsteiner, S, Vasserot, AP, Yaswen, P, hTERT expression can induce
resistance to TGFß growth inhibition in p16INK4A(-)
human mammary epithelial cells, Proc Natl Acad Sci (USA) 98:4498-4503, 2001. PMID:
11287649
Olsen CL, Gardie, B, Yaswen, P, Stampfer, MR,
Raf-1-induced growth arrest in human mammary epithelial cells is
p16-independent and is overcome in immortal cells during conversion, Oncogene
21:6328-6339, 2002. PMID: 12214273
Stampfer, M, Garbe, J, Nijjar, T, Wigington, D, Swisshelm, K, Yaswen, P, Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines, Oncogene 22:5238-5251, 2003. PMID: 12917625
Chin, K, Ortiz de Solorzano, C, Knowles, D, Jones, A, Chou,W, Rodriguez, E, Kuo, W-L, Ljung, B-M, Chew, K, Krig, S, Garbe, J, Stampfer, M, Yaswen, P, Gray, JW, Lockett, SJ. In Situ Analysis of Genome Instability in Breast Cancer. Nature Genetics:36, 984-988 2004. PMID: 15300252
Li Y, Pan J, Li J-L, Lee J-H, Tunkey C, Saraf K, Garbe J, Jelinsky S, Stampfer MR, Haney, SA, Transcriptional Changes Associated with Breast Cancer Occur as Normal Human Mammary Epithelial Cells Overcome Senescence Barriers and Become Immortalized. Mol Can 6:7, 2007. PMID: 17233903
Garbe, J, Holst, CR, Bassett, E, Tlsty, T, Stampfer, MR, Inactivation of p53 Function in Cultured Human Mammary Epithelial Cells Turns the Telomere-Length Dependent Senescence Barrier from Agonescence into Crisis. Cell Cycle 6:1927-1936, 2007. PMID: 17671422
Novak, P, Jensen, TJ, Garbe, JC, Stampfer, MR, Futscher, BW, Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization, Cancer Res 69:5251-58, 2009. PMID: 18922938
1. Pre-stasis (Chart
1, Panel C)
2. Post-stasis: Extended Life (EL) (p16
silenced/mutated) (Chart 1, Panel A)
Reduction
mammoplasty derived HMEC from specimen 184 that were exposed to the chemical
carcinogen benzo(a)pyrene in primary culture.
3. Post-stasis: Post-selection (p16 silenced) (Chart 1, Panel B)
Reduction
mammoplasty, non-tumor mastectomy, or benign tumor derived HMEC, grown in a
serum-free medium, that emerge from populations at stasis following silencing
of the p16 promoter.
4. Post-stasis: GSE22 (p53 expression inhibited by GSE22)
(Chart 1, Panel A)
Pre-stasis
HMEC grown in MM that were exposed to GSE22.
5. Post-stasis: p16sh (p16 expression inhibited by shRNA
to p16) (Chart 1, Panel C)
Pre-stasis
HMEC grown in a serum-containing medium that were exposed to shRNA to p16 and
show uniform bypass of stasis.
B.
FINITE LIFESPAN HUMAN MAMMARY FIBROBLAST CELLS
(HMFC)
D.
IMMORTALLY TRANSFORMED CELL LINES
1.
Immortal lines derived from cells grown in MM, following exposure to oncogenic
agents (Chart 1, Panel A)
1a)
Lines derived from cells exposed to benzo(a)pyrene (BaP); i.e. from the
post-stasis EL cultures
184A1
184A1-RF
184A1-GSE22
184A1-hTERT(a)
184A1-hTERT(b)
184A1- E6; -E7; -T; -E1A
184AA4
184AA2
184AA3
184AA5-7
184AaGS1,2
184AaMY1-5
184AaZN1-3
184AaE6
184B5
184B5-erbB2
184B5ME
184BE1
184BEMY1
184CEMY1
1b)
Line derived from cells exposed to GSE22
184FGS1
1c)
Line derived from cells exposed to hTERT
184FTERT
2.
Immortal lines derived from post-selection HMEC grown in serum-free MCDB170 (Chart
1, Panel B)
2a)
Lines derived from post-selection HMEC transduced with c-myc and/or ZNF217
184MY1
184ZN4-7
184ZNMY3-N
2b)
Lines derived from post-selection HMEC transduced with hTERT
184BTERT; 48RTERT; 161HTERT
2c)
Line derived from post-selection HMEC transduced with HPV-E6
184-E6
3.
Immortal lines derived from cells grown in the less stressful serum-containing
M85/M87/M87A media ± oxytocin (X) (Chart 1, Panel C)
3a)
Lines derived from following transduction with c-myc and/or shRNA to p16
184FMY2
184Fp16s1
184Fp16sMY
3b)
Lines derived from following transduction with hTERT
184DTERT