Interactions of apoptosis, proliferation and host age in the regression of the mouse mammary preneoplasia, TM3, carrying an unusual mutation in p53

Sharon G. Bonnette, Frances S. Kittrell, L.Clifton Stephens1, Raymond E. Meyn2 and Daniel Medina3

Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza,
1 Department of Veterinary Medicine and Surgery and
2 Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have developed an in vivo model system of mouse mammary preneoplasias in order to examine the cell and molecular changes that occur during tumorigenesis. Most of these preneoplasias are characterized by an alveolar hyperplasia morphologically similar to that present in normal pregnant mammary gland, but have tumor forming capabilities ranging from very low to high. One of these hyperplasias, the TM3 HOG (transformed mammary hyperplastic outgrowth), forms tumors infrequently and has the unusual characteristic of spontaneous regression. We have observed that 7–8 months post-transplantation into the cleared mammary fat pad of a BALB/c mouse, the TM3 hyperplasia will begin to regress, leaving only a sparse ductal tree with remnant alveolar structures by 10–12 months post-transplantation. We have sought to elucidate the mechanism of this regression by determining the apoptotic and proliferative rates of the alveolar cells during TM3 HOG development. Studies show that apoptotic rates in the TM3 HOG are consistently high (4–7%) at all times after transplantation. This apoptotic rate is higher than the rates found in other preneoplasias in our system and approach the rates observed in the normal involuting gland. An unusual p53 mutation, a serine insertion at codon 233, may be causally related to the high spontaneous apoptotic frequencies as well as elevated inducible apoptotic frequencies in TM3. In addition, a sudden decrease (~63%) in proliferation occurs around 8 months post-transplantation. Furthermore, transplantation experiments indicate that the ability of the 8-month-old host and/or mammary gland to support growth of preneoplastic mammary tissues is markedly diminished compared with 3- or 6-month-old hosts. The results presented here suggest that the persistent high apoptotic rates, concomitant with decreased proliferation rates, may be responsible for TM3's regression and implicate a unique mutant p53 as a causal factor. Additionally, the results suggest that host determinants can interact with specific molecular changes in the preneoplastic cells to influence growth and progression of the preneoplastic populations.

Abbreviations: AI, apoptotic index; TM HOG, transformed mammary hyperplastic outgrowth.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Because of the accessibility and experimental manipulability of the rodent mammary fat pad, the mouse mammary gland has been used extensively to study both normal and tumorigenic mammary development. We have reported the generation of a series of mouse outgrowth lines [EL ductal outgrowths and transformed mammary hyperplastic outgrowths (TM HOGs)] that can be perpetuated by serial transplantation into the cleared mouse mammary fat pad (1,2). The TM series of hyperplastic alveolar outgrowths exhibit both low and high tumorigenic potentials and are considered to represent cell populations at different stages in a progressing scheme of multi-step mammary tumorigenesis (3). For example, the TM3 HOG represents a slow-growing, ovarian-hormone-dependent alveolar preneoplasia with low tumorigenic potential [low tumor incidence (<20%) and a long tumor latency period (>12 months)]; in contrast, the TM2H HOG represents a fast-growing, ovarian-hormone-independent alveolar hyperplasia with high tumorigenic potential [a high tumor incidence (>90%) and a short tumor latency period (4 months)].

The TM3 outgrowth is particularly interesting and unique. Of the more than 35 preneoplastic outgrowth lines characterized by this laboratory over the past years, the TM3 line is the only line that spontaneously regresses as the host animal ages (3,4). The TM3 outgrowth line is extremely sensitive to ovariectomy and shows both decreased growth and loss of alveolar differentiation in hormone-deficient hosts (3). TM3 outgrowths, which are maintained in intact mice for 10–12 months, exhibit a loss of alveolar structures resulting in a sparse ductal outgrowth (3). We examined the proliferation and apoptotic characteristics of the TM3 outgrowths in order to understand the biological and cellular basis for the regressed phenotype.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
All recipient mice were inbred BALB/c, born and maintained in a closed conventional animal facility at Baylor College of Medicine, Houston, TX. The mice were fed Purina Rodent Blox ad libitum and maintained at 21–22°C with a 12 h light–dark cycle.

Mammary epithelium transplantation
Preneoplastic outgrowths were maintained by serial transplantation at 8–10 week intervals in the cleared mammary fat pads of 3-week-old female BALB/c mice as described by Kittrell et al. (1). For the experiments that examined growth potential of the preneoplastic outgrowths in adult mice, the mammary fat pads were cleared at 3 weeks of age and the mice not utilized for transplantation until they were either 8, 26 or 31 weeks of age. The transplants were prepared as whole mounts at 8 weeks after transplantation (3).

TUNEL analysis for apoptosis
The TACS 2 TdT in situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD) was used to detect apoptotic cells in mammary gland paraffin sections. Mammary glands were dissected and fixed immediately in neutral buffered formalin (100 ml formaldehyde, 900 ml dH2O, 4 g NaH2PO4, 6.5 g Na2HPO4) for 6–8 h at room temperature. The tissue was then rinsed twice in tap water and left in 70% ethanol until embedding and sectioning. The slides were deparaffinized and labeled according to the manufacturer's suggested protocol. A total of 500 cells were counted on each slide to determine the proportion of TUNEL-positive cells and generate a labeling index.

Morphometric analysis for apoptosis
BALB/c mice bearing preneoplastic outgrowths 11 weeks after transplantation were irradiated with a whole-body dose of 5 Gy using a 137Cs animal irradiator. Three mice were killed 6 h after irradiation. The rationale for sampling the tissues 6 h after irradiation was based on the results of prior studies demonstrating that the peak apoptotic response occurs at 6 h (5). The mammary glands containing the transplanted outgrowths were removed and prepared for histological sectioning. Tissues were embedded in paraffin blocks from which 4 µm sections were cut and stained with hematoxylin and eosin (H&E). Apoptotic cells were identified by morphological criteria described and illustrated previously (5,6). Apoptosis was scored by microscopic examination (at 400x) of H&E-stained sections on coded slides. Five fields were selected randomly in each specimen, and in each field the apoptotic cells in mammary ducts per 100 nuclei scored were recorded and expressed as a percentage [the apoptotic index (AI)]. The AIs reported are therefore based on scoring 500 nuclei for each specimen. Each AI represents an average for all specimens within that group. Differences in AIs between the groups were tested for statistical significance using the two-sided Student's t-test. Results were considered significantly different at P < 0.05.

PCNA immunohistochemistry
Fisher's microprobe system (Fisher, Pittsburgh, PA) was used for immunohistochemical staining. Paraffin-embedded sections were dewaxed by dipping in hemo D:xylene (3:1) twice for 2 min each and 100% ethanol once for 1 min. The samples were rehydrated by dipping twice in TTBS (0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, 0.01% Tween-20). Sections were blocked with 10% normal horse serum in TBS (0.1 M Tris–HCl pH 8.0, 0.15 M NaCl) for 30 min at 40°C. The serum block was removed and the section incubated with primary antibody (anti-PCNA 1:50; Santa Cruz, CA) for 15 min at 40°C. The sections were washed three times in TTBS for 1 min each at room temperature, and secondary antibody (goat anti-mouse biotinylated 1:2000; Vector, Burlingame, CA) applied for 15 min at 40°C. The sections were washed three times in TTBS and once in TBS only. ABC reagent (Vector) was applied to the sections for 12 min at room temperature. This was followed by a TBS rinse for 1 min and diaminobenzadine (DAB) staining. DAB reagent (Vector) was applied and incubated for 8 min. A TBS and dH20 wash immediately followed for 1 min each. The sections were counter-stained with 1% methyl green for 30 s and dehydrated, mounted and coverslips added. To calculate labeling indices, for each section, 500 cells were counted in two separate areas of the slide.

BrdU immunohistochemistry
Mice were injected i.p. with a 1:1000 dilution of Amersham Labeling Reagent (BrdU). Two hours later, animals were killed and the mammary gland tissue was sampled and put immediately into methacarn fixative (methanol:chloroform:glacial actetic acid, 60:30:10). The tissue was fixed overnight at room temperature and then switched to acetone until embedding in paraffin and sectioning onto Pro-Bond Plus slides (Fisher). The Amersham Cell Proliferation kit (Amersham, Arlington Heights, IL) was used for BrdU detection. The protocol was slightly modified by changing the primary anti-BrdU antibody incubation to 1.5 h at 42°C and the secondary peroxidase anti-mouse IgG2a antibody incubation to 1 h at room temperature. The sections were lightly counterstained with H&E. A total of 500 cells were counted on each section to calculate the percentage of cells labeled.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The TM3 outgrowth has low tumor-producing capabilities and begins to regress at 8 months post-transplantation
Table IGo shows the tumor incidences and latencies for the TM3 HOG and TM3 HOG sublines. The original TM3 was observed to be completely non-tumorigenic up until transplant generation 17 (TG17). The formation of random tumors gave rise to the establishment of the TM3-1 and TM3-2 hyperplastic outgrowth sublines in an attempt to maintain a subline which was non-tumorigenic. However, both sublines exhibited tumor capabilities of 13.6% for TM3-1 and 22.0% for TM3-2 at 12 months after transplantation. These tumorigenic capabilities are considered low compared with other TM preneoplasias in our system (3). Both TM3 sublines still exhibited growth characteristics similar to the parental TM3 outgrowth. Three months post-transplantation, the TM3 HOG filled 50–80% of the fat pad and exhibited a dense alveolar morphology (Figure 1AGo). Around 8 months post-transplantation, the alveoli of the outgrowth began to regress and this process continued until a complete regression of the hyperplastic outgrowth was obtained at 10–12 months. The regressed TM3 HOG exhibited a fine ductular structure with remnant alveoli (Figure 1BGo). Histological examination of the TM3 outgrowth also indicated a decrease of alveolar structures 8 months post-transplantation (Figure 2BGo) and a complete loss by 10 months (Figure 2CGo). This regression property was observed among all of the TM3 outgrowth lines.


View this table:
[in this window]
[in a new window]
 
Table I. TM3 sublines, transplant and tumor history
 



View larger version (219K):
[in this window]
[in a new window]
 
Fig. 1. TM3 whole mounts (magnification 6x). (A) TM3 hyperplastic outgrowth 3 months post-transplantation exhibiting a dense alveolar morphology that filled 46% of the fat pad. (B) TM3 hyperplastic outgrowth 10 months post-transplantation exhibiting an almost complete loss of alveolar structures leaving only a fine ductal morphology.

 


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. TM3 histology. The development of TM3 was scored by judging the alveolar density of the hyperplasia (magnification 100x). (A) Mature 4- to 6-month-old outgrowth displaying dense alveolar structures and would be given a score of `3'. (B) Seven- to eight-month-old outgrowth displaying characteristics of active regression. Alveoli in some parts of the gland are becoming sparse (arrow) while there are still parts of the gland which are of a medium density. This outgrowth would be given a score of `2'. (C) Eight- to ten-month-old gland with a TM3 outgrowth that has totally regressed, showing only remnant alveolar structures and remaining ducts (arrow). This outgrowth would be given a score of `1'.

 
Decreased rates of proliferation in the presence of high apoptotic rates result in TM3 regression
The normal involuting mammary gland exhibits a large induction of apoptosis after cessation of lactation that causes the loss of alveolar cells. Thus, one of our early hypotheses to explain TM3 regression was that the loss of alveoli in the TM3 hyperplasia follows the paradigm of normal involuting mammary gland and exhibits an induction of programmed cell death during regression. Analyses of apoptosis by the TUNEL method in random TM3 outgrowths between 4 and 7 months of age revealed that TM3 exhibited a consistent high apoptotic index of 4.6%, a level that approached those observed in normal involuting gland (7) (Table IIGo). Analyses of the cell cycling population by PCNA immunohistochemistry experiments showed an average of 30.6% labeled cells for all samples (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. Apoptotic indices of TM3
 
The samples analyzed in Table IIGo represented outgrowths from different donors and of different transplant generations. Since consistent changes in cell death or proliferation were not detected at these early time periods, a more systematic approach examined the apoptotic and proliferative rates of TM3 outgrowths during the early time periods of regression. Additional 7 and 8 month outgrowths from one subline (TM3-1, TG27) were harvested and analyzed by TUNEL and by BrdU labeling to determine apoptotic and proliferative indices. In these experiments, all of the TM3 transplants were from the same donor and transplantation was performed at the same time. The outgrowths were collected at precisely 7 and 8 months post-transplantation, the period when we could first detect morphological changes indicative of regression. Additionally, BrdU immunohistochemistry was used to obtain a more precise analysis of cells undergoing DNA synthesis. Results of these studies showed that 7-month-old outgrowths exhibited apoptotic indices of 7.2% and BrdU labeling indices of 14.4%, whereas the 8-month old outgrowths exhibited statistically similar apoptotic rates (5.5%) and a 63% reduction (P < 0.05) in BrdU labeling indices (5.3%) (Table IIIGo). Histological examination of the outgrowths revealed that the 8-month-old outgrowths were in the regression mode and exhibited sparse regressing alveoli versus the more dense alveolar structures of the 7-month-old outgrowths. These results suggested that the decreased proliferation concomitant with a relatively high apoptotic index in the 8-month-old outgrowths was responsible for their regression.


View this table:
[in this window]
[in a new window]
 
Table III. BrdU and TUNEL indices of 7 and 8 month TM3-1
 
It was puzzling that proliferation rates appeared to decrease between 7 and 8 months of age. To determine if this cellular response was unique to TM3 or perhaps due to a host-mediated effect on the growth of preneoplastic hyperplasias in general, we transplanted three different preneoplastic hyperplastic outgrowth lines with different biological properties and tumorigenic capabilities into the cleared fat pads of different aged mice (Table IVGo) and examined the extent of growth by whole mount analysis at 8 weeks after transplantation. To our surprise, the growth potential of all three preneoplastic hyperplasias was reduced by at least 50% when transplanted into 8-month-old mice compared with 3-week-, 4-month- or 6-month-old mice. Thus, host age had a significant impact on proliferation of the hyperplasias.


View this table:
[in this window]
[in a new window]
 
Table IV. The effects of age on TM preneoplastic growth
 
Apoptotic rates of the TM3 hyperplasia are highly induced in response to {gamma} irradiation
Because of a unique mutant p53 in TM3 outgrowths and the increased apoptotic frequency in these outgrowths, we tested the functional properties of the mutant p53 by examining radiation-induced apoptosis. Previous experiments had identified a mutant p53 (Ser233–234 insertion) in the TM3 hyperplasias and resulting tumors (8). Additionally, previous experiments demonstrated a close relationship between the presence of wild-type p53 and radiation-induced apoptosis (5). Figure 3Go illustrates the apoptotic frequency in TM3 outgrowths at 6 h after 5 Gy whole body irradiation of the hosts. Apoptotic nuclei were identified in H&E-stained mammary gland sections by morphometric analysis. The percent apoptosis in the TM3 is compared against normal pregnant gland, two other hyperplastic outgrowths (TM12, TM2L) that have wild-type p53 and two hyperplastic outgrowths (TM2H, TM4) that have other mutations in p53. The apoptotic response in irradiated TM3 was 2- to 2.5-fold greater than the response observed in the normal mammary gland and the hyperplasias exhibiting wild-type p53, and was clearly distinct from the absence of apoptosis observed in the hyperplasias containing either null (TM2H) or dominant-negative (TM4) p53 mutants. The presence of the mutant p53 thus provided one possible reason for the persistently elevated apoptotic activity in the TM3 cell population.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. {gamma}-Irradiation induced apoptosis in the TM3 HOG. The frequency of apoptosis (means ± SEM) is plotted for normal pregnant gland and five preneoplastic outgrowths that differ in p53 status. The frequency of apoptosis was determined by the morphometric assay described in Materials and methods, and by Medina et al. (5). The p53 status is stated at the top of the figure. The apoptosis frequency for TM12, TM2L, TM2H and TM4 was originally presented by Medina et al. (5).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The morphological and functional properties of theTM3 hyperplastic outgrowth have been described by Medina et al. (3). In these studies, the TM3 hyperplasias were of transplant generations (TG) 4, 5 and 6. Current transplant generations are around TG34, and the TM3 outgrowths reported in these studies were between TG24 and TG29. To date, the morphological and growth characteristics of the TM3 hyperplasias are essentially the same as the original. The TM3 line was first described as a slow-growing hyperplasia that exhibited a dense alveolar phenotype; however, the outgrowth filled only 50–75% of the fat pad and then stopped growing (3). It was reported to be completely non-tumorigenic having never given rise to a neoplasia (no tumors out of 114 transplants) unlike its other TM counterparts with tumor latencies that ranged from 4 (TM2H) to 11 months (TM10) and tumor incidences of 90 and 27%, respectively (3). Only recently, beginning at TG17, have random tumors arisen from the TM3 outgrowth. Although some tumors have recently been observed, the frequency is still low compared with the other TM lines of our system, and the basic growth and morphological characteristics are still the same as those in the previous studies (3). An additional characteristic that has remained unchanged from the earlier studies is the regression phenotype of the TM3. TM3 outgrowths that are maintained for >8 months in a virgin host animal are observed to undergo a regression of the alveolar phenotype (at 8 months), leaving a sparse ductal tree with only remnant alveoli. In addition to its low tumor-producing capabilities, the mechanism for this spontaneous, yet precisely timed event, has become the central interest of the TM3 hyperplasia.

There is evidence to indicate that apoptotic rates are regulated throughout mammary gland development and play a role in gland development. Five- to 6-week-old virgin BALB/c mice exhibit high levels of apoptosis (11.3%) in the terminal end buds of the mammary gland (9). The spatial restriction of apoptosis to the terminal end buds suggests a possible role for apoptosis in ductal formation (9). Additionally, the early to midpregnant mammary gland (<=13 days) exhibits significant amount (~3%) of apoptosis (6). In contrast, mature virgin mice, 17-day-old pregnant mice and lactating mice exhibit indices of 0.0, 0.2 and 0.0%, respectively (9,10). However, at 2–3 days of involution, apoptotic levels increase to 1.7% and by day 4 of involution reach peak levels of 4.5% (7).

We wanted to determine if regression of the TM3 hyperplasia followed the paradigm of normal mammary gland involution after lactation and weaning. If this were the case, we would have expected to see low levels of apoptosis during the actively expanding stage of TM3 and higher induced levels during the time of regression. These studies showed that the TM3 displayed relatively high levels of apoptosis (4.6%) between 4 and 7 months post-transplantation as compared with the other TM hyperplasias in our system (<=2%) (5), and the levels were very near those of the normal involuting gland. However, these studies did not show any changes in the apoptotic indices during the predicted time of regression. In contrast, we observed a dramatic decrease (63%) in the rate of cell proliferation in the 8 month TM3. We interpret these results to indicate that the marked change in the proliferation/apoptosis ratio from 2.0 to 1.04 was sufficient to cause alveolar cell loss.

Two early hypotheses proposed as mechanisms to explain the spontaneous regression of the TM3 hyperplastic outgrowth included a host-mediated immune response and an induction of alveolar cell death. Early descriptions of TM3 reported that there was only minor lymphocytic infiltration among the epithelial cells of the regressing gland, suggesting that an immune response was not likely to be responsible for the loss of epithelial cells in the regressing TM3 (3). To confirm this, we performed a classical immunological challenge protocol by transplanting TM3 into TM3-challenged and -unchallenged hosts while controlling for the age of the hosts (unpublished data). This experiment was uninformative as to an immunological effect because the TM3 transplants had similar, but suppressed, growth capabilities in both the 8-month-old challenged and unchallenged hosts. Any suggestion of a host immune response could have been masked by TM3's limited growth in an aged mouse. The effect of host age on net growth was demonstrated to be a general phenomenon, as two other preneoplastic hyperplasias, TM2H and TM2L, exhibited a 50% lower net growth in 30- to 36-week-old mice than in 3- or 26-week-old mice. Interestingly, neither the TM2H nor the TM2L hyperplasias regressed in the 8-month-old mice.

The reasons for the decreased growth potential that resulted in regression of the TM3 hyperplasia in older animals may be due to a couple of mechanisms: (i) a decreased hormonal environment and (ii) alterations in the local stromal microenvironment due to aging. Estrogen and prolactin are hormones important to mouse mammary gland development and tumorigenesis (11,12). The TM3 hyperplasia has been shown to be extremely dependent on ovarian hormones as demonstrated by its decreased growth rate and loss of alveolarity in ovariectomized host (3). Thus, it is reasonable to suggest that the decreased proliferation and subsequent regression of TM3 might be caused by changes in hormone (e.g. estrogen, prolactin) levels in adult mice; however, the maintenance at high fertility until at least 12 months of age does not indicate significant hormonal changes. Surprisingly, there are no reports available that address the changes in ovarian hormone levels in mid-adult life of the BALB/c strain of mice. Moreover, enhanced hormonal secretion by pituitary isografts that result in persistent lobuloalveolar development of the normal mammary gland did not prevent the spontaneous regression of the TM3 outgrowths (unpublished observation). Therefore, at this time there is little direct evidence to support this mechanism.

Little is known about the role of the mammary fat pad in mammary cell growth and morphogenesis. There is evidence to suggest that the epithelium communicates with the stroma to regulate the activities of the stromal compartment and that the stroma has influence on the activities of the epithelium (13). Stromal derived factors that may influence the activity of mammary epithelium include HGF/SF, heregulin, IGF-I and -II, FGF-2 and -7, TGF-{alpha}, and Wnt-2, -5a and -6 (13). It is still not known what the absolute functions of many of these factors are in terms of stromal-mediated paracrine effects on mammary epithelium. Further information on stromal and epithelial contributions to mammary gland development will require tissue-specific knockout models or the use of stromal-specific promoters to target overexpression solely to the adipose or fibrous stroma. To address the question of the effects of aging on the mammary fat pad, whole fat pad transplants of different ages into different aged hosts can be performed to test whether TM3 regression and compromised growth in the 7 month or older mice is completely fat pad dependent. The transplant studies are long-term experiments but they can provide clues to the growth factor requirements and the role of the fat pad microenvironment in both normal and neoplastic growth: In preliminary experiments, we examined the ability of young (3-week-old) and aged (43-week-old) mammary fat pads to support growth of TM2L hyperplastic outgrowth line in young and aged host environment. The young fat pads support growth equally well in hosts of either age, but the old fat pad supports growth in a young environment but not in the aged environment. This result suggests there is a complex set of interactions between host systemic factors and the aged fat pad (D.Medina, unpublished data).

The possibility that the mutation in p53 specific to the TM3 cells (Ser233–234) is the molecular mediator for increased apoptosis levels is intriguing and supported by the irradiation data. Previous reports lend support to this idea. Li et al. (14) have reported the generation of a transgenic mouse line that preferentially overexpresses a murine 172Arg–Leu mutant p53 under the control of the whey-acidic protein promoter. These mice exhibit high levels of mammary gland apoptosis (~20%) at day 17 of pregnancy compared with their non-transgenic counterparts (~0.2%) and, in response to irradiation, 15-day-old pregnant glands exhibit twice the induction levels that the wild type glands experience (10,14). Additionally, the p53-mediated apoptosis in these transgenic mice is observed to delay 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis (10). The high basal levels of apoptosis, the enhanced apoptotic response to irradiation and the reduced susceptibility to tumorigenesis in the 172Arg–Leu mutants are reminiscent of characteristics observed for the TM3 hyperplasia. The TM3 mutant p53 exhibits properties similar to those observed for the 172Arg–Leu transgene with regard to enhanced apoptotic response to irradiation, and the TM3 preneoplastic outgrowth is highly resistant to DMBA-induced tumorigenesis (D.Medina, unpublished data); however, it is not presently known whether the Ser233–234 mutant p53 possesses wild-type trans-activating properties. By acting as a `super' p53, the Ser233–234 mutation may be responsible for TM3's relatively slow growth, high spontaneous and inducible apoptotic rates and protection from tumor formation.

Interest in the biological processes underlying tumorigenic capability has focused on factors regulating proliferation; however, a better understanding of regression may provide additional insight and uncover other important mechanisms. Spontaneous regression of malignancy is rare, and when it does occur, the mechanisms surrounding the regression remain completely unknown (15). In some cases, host defense mechanisms are implicated as a source of the regression (16); however, in the TM3 case, there is little evidence for this mechanism. Although the TM3 is viewed as a weakly tumorigenic cell population, understanding the mechanisms that underlie TM3 regression could provide valuable information for the generation of preventative and therapeutic strategies.


    Acknowledgments
 
We gratefully acknowledge the technical assistance of Elizabeth Hopkins for preparation of the histological sections. These studies were supported by grants CA 25215, CA 47112, DAMD 17-94-J4204 (D.M.) and CA 69003 (R.E.M.).


    Notes
 
3 To whom correspondence should be addressed Email: dmedina{at}bcm.tmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Kittrell,F.S., Oborn,C.J. and Medina,D. (1992) Development of mammary preneoplasias in vivo from mouse mammary epithelial cell lines in vitro. Cancer Res., 52, 1924–1932.[Abstract]
  2. Medina,D. and Kittrell,F.S. (1993) Immortalization phenotype dissociated from the preneoplastic phenotype in mouse mammary epithelial outgrowths in vivo. Carcinogenesis, 14, 25–28.[Abstract]
  3. Medina,D., Kittrell,F.S., Liu,Y. and Schwartz,M. (1993) Morphological and functional properties of TM preneoplastic mammary outgrowths. Cancer Res., 53, 663–667.[Abstract]
  4. Medina,D. (1996) Preneoplasia in mammary tumorigenesis. In Dickson,R.B. and Lippman,M.E. (eds) Mammary Tumor Cell Cycle, Differentiation and Metastasis. Norwell: Kluwer Academic Publishers, Boston, pp. 37–69.
  5. Medina,D., Stephens,L.C., Bonilla,P.J., Hollmann,C.A., Schwahn,D., Kupperwasser,C., Jerry,D.J., Butel,J.S. and Meyn,R.E. (1998) Radiation-induced tumorigenesis in preneoplastic mouse mammary glands in vivo: Significance of p53 status and apoptosis. Mol. Carcinog., 22, 199–207.[ISI][Medline]
  6. Meyn,R.E., Stephens,L.C., Mason,K.A. and Medina,D. (1996) Radiation-induced apoptosis in normal and pre-neoplastic mammary glands in vivo: significance of gland differentiation and p53 status. Int. J. Cancer, 65, 466–472.[ISI][Medline]
  7. Quarrie,L.H., Addey,C.V. and Wilde,C.J. (1995) Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labeling. Cell Tissue Res., 281, 413–419.[ISI][Medline]
  8. Jerry,D.J., Ozbun,M.A., Kittrell,F.S., Lane,D.P., Medina,D. and Butel,J.S. (1993) Mutations in p53 are frequent in the preneoplastic stage of mouse mammary tumor development. Cancer Res., 53, 3374–3381.[Abstract]
  9. Humphreys,R.C., Krajewksa,M., Krnacik,S., Jaeger,R., Weiher,H., Krajewski,S., Reed,J.C. and Rosen,J.M. (1996) Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development, 122, 4013–4022.[Abstract/Free Full Text]
  10. Li,B., Kittrell,F.S., Medina,D. and Rosen,J.M. (1995) Delay of dimethylbenz[a]anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein. Mol. Carcinog., 14, 75–83.[ISI][Medline]
  11. Imagawa,W., Bandyopadhyay,G.K. and Nandi,S. (1990) Regulation of mammary epithelial cell growth in mice and rats. Endocrine Res., 11, 494–523.
  12. Snedeker,S.M. and Diaugustine,R.P. (1996) Hormonal and environmental factors affecting cell proliferation and neoplasia in the mammary gland. In Huff,J., Boyd,J. and Barrett,J.C. (eds) Cellular and Molecular Mechanisms of Hormonal Carcinogenesis: Environmental Influences. Wiley-Liss, New York, pp. 211–253.
  13. Neville,M.C., Medina,D., Monks,J. and Hovey,R.C. (1998) The mammary fat pad. J. Mamm. Gland Biol. Neoplasia, 3, 109–116.[ISI][Medline]
  14. Li,B., Greenberg,N., Stephens,L.C., Meyn,R.E., Medina,D. and Rosen,J.M. (1994) Preferential overexpression of a 172Arg-Leu mutant p53 in the mammary gland of transgenic mice results in altered lobuloalveolar development. Cell Growth Differ., 5, 711–721.[Abstract]
  15. Kaiser,H.E. (1994) Biological viewpoints of neoplastic regression. In Vivo, 8, 155–165.[Medline]
  16. Lattouf,A.N., Rizk,A.T. and Gedeon,E.M. (1994) Spontaneous regression of malignant lymphoma. Case report and review of the literature. Lebanese Med. J., 42, 37–38.
Received March 16, 1999; revised May 13, 1999; accepted May 14, 1999.