HUMAN MAMMARY EPITHELIAL CELL NEWSLETTER #2 September 1988

Introduction

In this newsletter, I want to go over the derivation of the immortalized cell lines 184A1 and 184B5, and the numerous subclones and variants that have been isolated from these lines. The aim is to familiarize others with what cell types are available, how they were obtained, and what is a uniform nomenclature for these cells.

Extended Life (EL) Cultures

The origins of the cell lines are described in our PNAS paper [1], and more detail about the extended life (EL) cultures which preceeded the lines is presented in two recent review articles [2,3], so I'll not repeat most of that information. Basically, I took out frozen ampoules of 184 organoids on three separate occasions. The three separate ampoules were given the 3 different Freeze-down symbols (see first newsletter) of "aleph"(A), "cross"(C), and "birdie"(B). In each case, at least 2 T-25s were seeded and treated as controls, and 2 T-25s were treated for 2 or 3 24hr periods with 1ęg/ml of benzo(a)pyrene. The cells were being fed with our MM medium, in which 184 grows rapidly in primary culture and normally senesces by 5th passage. We followed the fate of the treated and control cells both in primary culture (how long they maintained growth) and after subculture (the primary flasks were partially trypsinized many times, and some of the removed cells plated for subculture). Figure 1 (which is in references 2 & 3) shows the fate of these cells. I show it here to go over some nomenclature points. We gave numerical labels to each of the followed subcultures in experiments C (cross) and B (birdie); i.e., the B1, B2 B3 etc. that you see in the figure. The EL cultures (i.e., the treated cells that kept growing after the controls had fully senesced) derived from each of these subcultures were given the alphabetical equivalent to these numbers, e.g., B2 = Bb, C5 = Ce. The immortally transformed lines that developed were given the subculture name, i.e., B5 and A1. We have limited frozen stocks for some of these EL cultures. These stocks are made possible by transferring the very small amounts of MM grown frozen cells to MCDB170, which permits growth for an additional 2-5 passages. Since the cell amounts are limited we're not eager to send them out; however, if important, we can grow and fix cells here, or make limited amounts available. The following EL types are available:

Subclones Isolated from 184A1 and 184B5

Derivatives of the lines have been isolated by (1) cloning; (2) selection for growth in more stringent media; (3) exposure to a chemical carcinogen followed by selective growth conditions; (4) exposure to oncogenic viruses and oncogenes.

Our cloned isolates from 184A1 are referred to as A1L(number)(currently 2-8). In general, about 50-100 cells were seeded into 60 or 100mm dishes, and well defined, clearly isolated, good growing colonies picked with a cloning cylinder and seeded 1 each into 35mm dishes. Of 8 picked, 5 grew up sufficiently well to amplify and freeze down. Since A1 has a tendency to spread out as colonies, it is possible that these clones, which were aggressive growers in a smaller spacial area, may not be truly representative of the A1 mass population. We have the following: A1L2, seeded at 31 , first frozen at 35 ; A1L5, seeded at 28 , first frozen at 32, A1L6, A1L7, A1L8, seeded at 33 , first frozen at 38 , 37 , and 37 respectively.

We first isolated clones from 184B5 early in its history, since its well defined, colonial growth pattern made it easy to clone. At 13 , 50 cells were seeded into 35mm dishes. 4 colonies were isolated with cloning cylinders. They were picked for representing the extremes of the B5 morphology - 2 growing in even Tighter patches than the norm, and 2 growing in slighter Looser patches. Of these, 2 grew up, B5T1 and B5L3. These were first frozen at 16 . More recently, we isolated several more B5 clones. Approximately 50 cells were seeded into 35mm dishes at 25 . 2 colonies of tight morphology were picked and maintained a homogeneous tight morphology upon subculture (called B5T2, and B5T3). One picked colony looked heterogeneous and was recloned, yielding B5T4 and B5L4. These clones were first frozen at 29-31 , and we are currently still growing B5T2, B5T3, and B5L4 (now at passages 44, 37, and 43). With the exception of B5T1, the A1 and B5 clones present a relativly homogeneous morphology and grow consistantly for as long as they have been passaged. B5T1 has some strange properties. It's morpholgy tends to be more heterogenous - the tight patches of small packed cells also have larger, more "fried egg" looking cells present at all passages. In three separate instances, when the cells reached around 29 , they were observed to look poor ("yukky" & "gross" were the comments). Almost no patch showed growth past 30 ; however, the very rare patches that did keep growing yielded more homogeneous, good growing populations by 34 . The cell pools resulting from these 3 separate instances of survival through this "crisis" have been given the subtype names of B5T1 P1, P2, and P3. Unlike the parental B5 population, which shows only little growth inhibition in response to TGF- , the B5T1 cells prior to crisis are extremely and completely sensitive to TGF- growth inhibition.

Subpopulations Isolated by Growth in More Stringent Media

Early populations of A1 and B5 were grown in MM, and subpopulations were selected for growth in MM minus specific factors. Later, both lines were tranfered at early passage (11) to MCDB170, and subpopulations have been isolated that do not require specific growth factors present in MCBD170. In all cases where spontaneously occuring subpopulations have been isolated based on the nutritional composition of the medium, the nomenclature has been to call them A1N... and B5N... In the case of MM derived subpopulations, the N is followed by a number, in the case of MCDB170 derived cells, the N is followed by letters indicating what factors were absent.

Perhaps the most used of these subpopulations is A1N4 [see reference 4]. These cells were seeded at 16 in MM minus the conditioned media and without cholera toxin. Attachment appeared poor but the few patches that were present grew fairly well. After 3 passages, there appeared to be uniform good growth and attachment. These cells were first transferred to MCDB170 at 28 . The karyology of A1N4 indicated that unlike the near diploid A1, these cells were aneuploid, near triploid, but contained only one additional chromosomal marker beyond the 4 seen in the parental A1 cells [4]. It is likely then, that although they were not cloned, they represent a clonal population. Because they could grow in a simpler medium than MM or MCDB170, we gave these A1N4 cells to Robin Clark at Cetus for experiments on malignant transformation with oncogenes. We have frozen several other A1 and B5 populations from MM, but these have not been used for anything (e.g., A1N3 = minus hydrocortisone; A1N11 = minus conditioned media and EGF; B5N4 = minus conditioned media; B5N7 = minus conditioned media and FCS).

We later tested the growth requirements of 184A1 and 184B5, along with the parental 184 cells, in MCDB170 lacking individual growth factors. Some of these data can be found in reference 3. These first tests were done over the course of one passage, and so did not indicate if there were individual cells in the population which could maintain growth in the absence of the growth factors. We therefore grew mass cultures of 184, 184A1, and 184B5 for extended passages in MCDB170 lacking either insulin (I), EGF, bovine pituitary extract (BPE), or hydrocortisone (HC). Since these cells may be of value for certain kinds of studies, I'll go into some detail of their history:

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184: (experiments were started at 7th or 8th passage, with batches that senesced around 15 )
-EGF caused only a slight slowdown in growth rate, no noticeable morphological change; passage at senescence was one lower. Explainable by endogenous production of TGF-a.
-I caused a noticeable slowdown in growth rate, cells looked somewhat less healthy, but maintained growth until the same, or one lower, passage of senescence. -HC the cells looked sick and grew to confluence for only 1 or 2 passages.
-BPE gave very variable results with different cell batches and other possible unknown variables. Basically the cells would grow only for 1-3 passages.

184A1: (done once, media first changed at 27-32 )
-EGF (resultant population called 184A1NE). For the first 2 passages, growth was slow and selective (a small # of patches). The growth rate was the same as control (+EGF) after 4 passages.
-I (called 184A1NI). Growth was initially slowed, but less selective than -EGF; the cells looked good. Growth rates were normal within 2-4 passages.
-BPE (called 184A1NB). Little initial growth. Eventually a few patches grew out. After one additional passage the resultant cells grew normally.
-HC (called 184A1NH). Slow patchy growth for first passage; growth normal after 2 passages.

184B5:
-EGF (called 184B5NE). Media first changed at 48 . Growth initially slower and selective. It took 7 passages to select a population that looked good and had a normal growth rate. Repeating with cells at 36 , after 3 passages of slow, selective growth a good growing population seems to be arising.
-I (called 184B5NI). Media changed at 35 . Growth initially slowed but not as selective as -EGF. Growth rates were normal within 4-6 passages.
-BPE (called 184B5NB). Growth initially slow and selective but not as extreme as the A1 cells
-BPE. Cells didn't look good and grow normally until after 6 passages.
-BPE & I (called 184B5NIB). 184B5NB cells were switched at 42 to media without insulin. They grew initially slowly and poorly. Good patches were obvious after 2 passages and growth was normal after 3 passages.
-HC (called 184B5NH). Media changed at 34 . Growth initially slower but normal after 2 passages.

Subpopulations Isolated After Further Carcinogen Treatment

184A1 and 184B5 were exposed in separate experiments 2 or 3x to ENU (1500ęg/ml for A1, 750ęg/ml for B5). Treated and control cells were then seeded into stringent media (lacking combinations of growth factors) that we had already shown did not support normal A1 or B5 growth. I'm still in the process of sorting out all the resultant data. For general circulation, the following variants of potential interest have arisen (all carcinogen treated A1 and B5 cultures have a "Z" after their name; here the Z is followed by information about the nutritional selection):

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184A1ZNEB: selected in MCDB170 - EGF and BPE. The treated cells had a fair amount of growth (compared to almost nothing in the controls), but most of this faded away after several passages. In one experiment, cells with patchy vigourous growth and a distinctive morphology quickly took over the population, and have maintained active growth in this medium.

184B5ZNEI: selected in MCDB170 - EGF and I. Again the treated cells showed initially widespread, morphologically heterogenous growth (compared to very little in the controls). Again most of this growth faded after about 5 subcultures but in several cultures growth was maintained. The morphologies are not particularly distinctive and we don't know if these represent clonal cultures.

Subpopulations Isolated After Exposure to Oncogenes

Both 184A1 and 184B5 have been exposed to oncogenic viruses or oncogenes introduced via retroviral vectors. In some cases, tumorigenic variants have been produced. The following cell types currently exist:
184A1N4 exposed to Harvey sarcoma virus (184A1N4-ras or 184A1N4-H), SV40 T antigen (184A1N4-T), v-mos (184A1N4-mos or 184A1N4-M ), and combinations thereof (184A1N4-T-ras or 184A1N4-TH, 184A1N4-mos-ras or 184A1N4-MH ). See reference [4] for details. Use of these cells also requires permission from Cetus Corporation (see under Robin Clark in enclosed list of investigators).

184A1 and 184B5 were also exposed to Kirsten sarcoma virus with a baboon pseudotype. This virus alone led to some anchorage independent growth (0.1-0.2%), but not tumorigenicity in the A1 cells (184A1-K), and to tumorigenicity (100% of exposed animals), but not anchorage independent growth in the B5 cells (184B5-K)[2]. Two tumors were removed from the animals and regrown in culture (184B5-KTu1, 184B5-KTu2).

As further experiments are performed exposing these cells to oncogenes, we may need to take another look at this nomenclature. In my computer records (see newsletter #1), I file all virus exposed cells as type = name followed by V, and subtype = name of viruses, e.g., 184A1N4-TH would be type = A1N4V, subtype = SVT, HSV.

A further nomenclature note: these names get rather unwieldy, and in informal discussion tend to be abbreviated, e.g., A1, B5, T1, B5NI, N4, NEB etc. However, for publication purposes I would appreciate if everyone would use the complete nomenclature.

One final note: I have just been informed that the patent application for A1, B5 and their derivatives has been allowed and will be issued shortly. Those of you who have been sent forms for use of these cells and have not yet returned the forms should do so promptly. A1 and B5 have been deposited at ATCC and mechanisms will be arranged for direct aquisition from ATCC; this will still entail filing of an experimental plan.

References

1. 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.

2. Stampfer, M.R., and Bartley, J.C., Growth and Transformation of Human Mammary Epithelial Cells in Culture. In: Cellular and Molecular Biology of Mammary Cancer (D. Medina, W. Kidwell, G. Heppner, E. Anderson, eds.) Plenum Publishing, New York, NY, pp.419-436, 1987.

3. Stampfer, M.R., and Bartley, J.C., Human Mammary Epithelial Cells in Culture: Differentiation and Transformation. In: Breast Cancer: Cellular and Molecular Biology ( M. Lippman, R. Dickson, eds.) Kluwer Academic Publishers, Boston, MA, pp.1-24, 1988.

4. Clark, R., Stampfer, M., Milley, B., O'Rourke, E., Walen, K., Kriegler, M., Kopplin, J., and McCormick, F., Transformation of Human Mammmary Epithelial Cells by Oncogenic Retroviruses. Cancer Research 48:4689-4694, 1988.