Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.

Kupffer cells, which are part of the reticuloendothelial system, play an important role in clearing pathogenic substances, including tumor cells, from the liver. The role of Kupffer cells in tumor development is very important as Kupffer cells can be manipulated to a tumoricidal state with biological response modifiers to kill tumor cells and thus to decrease tumor burden and extend survival time. To gain additional information on the role of Kupffer cells and their interaction with tumor cells in hepatic metastases, we studied an established experimental hematogenous metastatic model (Friend erythroleukemia) in mouse livers by light and electron microscopy. Highly activated Kupffer cells were observed in close contact with tumor cells in sinusoids and also in tumor forming foci within the hepatic parenchyma. The Kupffer cells were activated by the presence of the hematogenous tumor cells and were able to lyse and phagocytose them. However, some tumor cells evaded the Kupffer cells as metastases still occurred. Kupffer cells and other macrophages were found to leave the sinusoids and migrate to sites of potential tumor development where they interacted with tumor cells and intimately wrapped their processes around fat storing cells. It is possible that these macrophages which cross biological barriers could be used to deliver drug-loaded microparticles (liposomes and microcapsules) to tumors.

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.

Virus-like particles were found in two transplantable tumours, Sp56 and Sp6, from BDX rats. Sp56, a neurogenic sarcoma, contains abundant C-type particles in all stadia nof morphogeneis. This tumour reacts with anti-Friend leukaemia virus gp70 and anti-Rauscher leukaemia virus p30 sera. Sp6, a fibrosarcoma, has abundant virus-like particles in the cytoplasm, very often associated with centrioles or basal bodies of a cilium. These particles consist of two concentric shells with a diam. of 60 to 65 nm. Released particles were found outside the cell with a diam. of 85 to 100 nm characterized by an envelope and an eccentrically located electron-dense nucleoid, surrounded by an intermediate layer. These virus-like particles show no cross-reaction with antisera against murine C- or B-type particles, but show ultrastructural similarity with virus particles recently described in Chinese hamster cells and in mouse cell lines infected with two retrovirus isolates from South-East Asian mice.

Serum thymic factor (STF) and azathioprine (AZ) inhibition test were assayed in sera and spleen cells from DBA/2 and BALB/c mice, inoculated with the anemic strain of Friend leukemia virus. The observed decrease of STF levels appears to be related to the LLV (Lymphatic Leukemia Virus) component of FLV-A.

In vivo infection of DBA/2 and D2.Fv-2r mice with 10(5) FFU of FV resulted in a rapid adsorption of virus onto the spleen cells. Thus, these cells could be used in the analysis of Fv-2 expression on FV target cells without any difficulty in avoiding extracellular FV particles. Higher frequencies of cells capable of forming IC could be detected in the spleen than in the bone marrow at 2 and 48 h post infection and levels were very low in D2.Fv-2r mice. When recently infected cells were transferred to secondary Fv-2s recipients shortly after infection had been established, the virus-replicating cells released SFFV equally well regardless of whether they originated from Fv-2s or Fv-2r mice. This suggests that suppression of SFFV replication may not take place in Fv-2r cells at an early stage of infection.

To test the ability of cloned committed erythroleukemic cells to participate in development we have injected. Friend leukemia cells (FLC) into C57Bl/6 mouse blastocysts together with Friend leukemia virus (FLV) and we have examined the newborn individuals derived from them. Five animals out of 32 born have FLC-derived neoplasia. The incidence of neoplasia is increased as compared with other similar experiments without the virus. In two of the animals with the FLC neoplasia the disease manifestation is an erythroid leukemia similar to the one obtained directly with the virus in normal DBA/2 mice.

Congenic and double congenic mice expressing different H-7 alleles with varying H-2 haplotypes on the C57BL/10 (B10) background were tested for their susceptibility to Friend virus (FV) infection. Mice expressing the H-7b allele derived from BALB/c were susceptible to FV infection whereas mice expressing the H-7a allele of B10 were absolutely resistant. Coexpression of H-7b and the H-2a and H-2d haplotypes determined high susceptibility; mice expressing both H-7b and H-2b were relatively resistant compared to H-7b mice expressing H-2a or H-2d. Parallel experiments with BALB/c and BALB.B recipients did not demonstrate differences in susceptibility associated with H-2d and H-2b. Mapping experiments with the H-2h4 and H-2i5 recombinants indicated that the gene determining relative resistance to FV-induced spleen focus formation mapped to the K-end (KbAb) of H-2b. Female recipients expressing H-7b, regardless of their H-2 haplotype, were more susceptible to FV infection than their syngeneic male counterparts. These experiments demonstrate the utility of congenic and double congenic strains that define H-7 and Fv-2 on different H-2 haplotype backgrounds for the analysis of Fv-2:H-7:H-2 interactions in determining susceptibility to FV infection.

Typical morphological features of surface structural alterations during Friend cells differentiation are described. Scanning electron microscopy (SEM) revealed that DMSO induction switched on cell alteration of the proerythroblast-like cells, possessing microvilli projections on cell membrane with some ruffles, to an advanced stage with a blebby surface. This was followed by the formation of a pear-like polarized cell separated into two zones by a narrow cytoplasmic bridge at the equatorial plane. The polarized cells showed a smooth surface and tended to disconnect into two unequal cells. The villous leukemic erythroblast has negatively charged sialic acid residues on the glycocalyx, available for latex hydrazide probe binding, while the blebby and polarized cells lack it. Tocopherol added to culture medium of DMSO-induced erythroleukemic cells prevented the formation of blebs and the polarization phenomena, without affecting hemoglobin synthesis. The tocopherol-treated cells contain available negative charges for latex hydrazide binding similar to uninduced Friend cells. Erythropoietin potentiated a repolarization ability and morphological alteration capacity to the tocopherol-treated cells and this was accompanied by a loss of glycocalyx-negative charges. At these growth conditions erythyropoietin induced a dose-dependent proliferation effect.

A potent tumour promoter on mouse skin, phorbol-9-myristate-9a-acetate, induces certain clones of Friend erythroleukemia cells to become adhesive to the surface of tissue culture dishes, whereas in the absence of this compound, these cells grow in suspension. We have quantitatively tested 20 other phorbol esters and related compounds for this effect. When the results are expressed as the concentrations of compounds which show half-maximum effect on cell adhesion, the decreasing order of potency is: phorbol-9-myristate-9a-acetate (3.6 x 10(-10) M) approximately equal to gnilatimacrin greater than milliamine A approximately equal to phorbol-9,9a-didecanoate approximately equal to mezerein approximately equal to gnidilatin approximately equal to ingenol-3,20-dibenzoate greater than phorbol-9-myristate-9a-acetate greater than phorbol-9,9a-dibutyrate approximately equal to phorbol-9-9a-dibenzoate greater than 4a-O-methyl-phorbol-9-myristate-9a-acetate greater than phorbol-9-myristate-9a-acetate-3-aldehyde greater than phorbol-9,9a-diacetate greater than 2,3-dihydrophorbol-9-myristate-9a-acetate. Phorbol, 4a alpha-phorbol-9,9a-didecanoate, phorbol-3,9,9a-triacetate, phorbol-9-myristate, phorbol-9-monoacetate and phorbol-9a-monoacetate were inactive in this assay when tested at concentrations as high as 1 microgram/ml (10(-6) M). None of these 20 compounds induced adhesion when they were tested with a variant clone of Friend erythroleukemia cells which is resistant to the induction of adhesion and several other effects of phorbol-myristate-acetate. When the relative potencies of these compounds in the adhesion assay were compared to available in vivo data on tumour promoting activity on mouse skin, there was, in general, a good qualitative correlation. A better but not perfect quantitative correlation was obtained when the results from the adhesion assay were compared with reported inflammatory activity on mouse ear. When several other tumour promoters and cocarcinogens which differ structurally from the phorbol esters and related plant diterpenes were tested, none of these induced adhesion in this assay.