Breast cancer manifests itself in the mammary epithelium, yet there is a growing recognition that mammary stromal cells also play an important role in tumorigenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer, and many of the stromal factors necessary for mammary development also promote or protect against breast cancer. Here we review our present knowledge of the specific factors and cell types that contribute to epithelial-stromal crosstalk during mammary development. To find cures for diseases like breast cancer that rely on epithelial-stromal crosstalk, we must understand how these different cell types communicate with each other.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G 1 -specific cell cycle arrest through induction of p21 Cip1 . This in turn leads to inhibition of cyclin D-and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracyclinedependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.The serine-threonine protein kinase Raf is an essential component of the signal transduction pathway leading to mitogenactivated protein (MAP) kinase activation by growth factors which work through Ras (for reviews see references 32 and 53). Raf is a direct downstream effector of Ras (58, 90, 91) and phosphorylates MAP kinase-extracellular signal-regulated kinase (ERK) (MEK) (10, 38), which in turn activates the p42 and p44 ERK kinases (1,25,37,60), resulting in their translocation into the nucleus (8,42). This cascade of events leads to the activation of transcription factors and eventually to cell proliferation (for reviews see references 52 and 88). Blocking endogenous raf mRNA expression with antisense constructs or dominant negative Raf mutants interferes with mitogen-induced proliferation of NIH 3T3 cells (30,36,77), while microinjection of bacterially expressed c-Raf-1 protein is sufficient to induce DNA synthesis in quiescent cells (81).Raf was first discovered as the transforming principle of avian and murine retroviruses (2, 34). Oncogenic activation of Raf can be achieved by truncation of its N-terminal regulatory domain (2, 33) or by constitutive translocation of Raf to the plasma membrane by adding a farnesylation signal, such as the CAAX motif (41). Activated Raf proteins readily transform established fibroblast cell lines and can confer growth factor independence to avian and murine macrophages (6, 26). Moreover, similar to Ras (39, 73), Raf cooperates with other cellular and viral oncogenes, such as the Myc and simian virus 40 large T antigen genes, and has been shown to be transforming in the absence of p53 function in various cell types (3,26,46,54).In addition to the notion that Raf signalling plays a k...

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

Mammary gland development is a complex process that is dependent on interactions between the developing mammary epithelium and the surrounding stromal tissues. We show that mice lacking the triglyceride synthesis enzyme acyl CoA:diacylglycerol transferase 1 (DGAT1) have impaired mammary gland development, characterized by decreased epithelial proliferation and alveolar development, and reduced expression of markers of functional differentiation. Transplantation studies demonstrate that the impaired development results from a deficiency of DGAT1 in both the stromal and epithelial tissues. Our findings are the first to link defects in stromal lipid metabolism to impaired mammary gland development.

We have constructed a series of reporter constructs which test the effects of sequence elements from the control region of human mitochondrial DNA on expression in the nucleus, as assayed by transient expression in cultured human cells. The mitochondrial heavy-strand promoter (HSP) was unable to function as a promoter in nuclear DNA. Neither the HSP, nor the binding region for the mitochondrial transcription factor mtTF1 from the light-strand promoter, had any significant or systematic modulatory effects upon transcription from strong or weak RNA polymerase II (pol II) promoters, in three different human cell lines. The same finding held true regardless of orientation with respect to the start site of transcription. Similar results were obtained with a rho 0 derivative of one of these lines, indicating that mitochondrial promoter sequences in the nucleus cannot modulate transcription in response to altered mtDNA copy number. These results support the view that the nuclear and mitochondrial transcription systems in human cells are functionally independent, and do not communicate through factors recognizing shared sequence elements, as suggested by studies in yeast.