Peter Alexander

The significance of macrophages in human and experimental tumors.

Macrophages are involved at several stages in the genesis of an immune response to tumors. They process antigens, they are required in the transformation by tumor antigens of memory lymphocytes into cytotoxic lymphocytes, and they are also important effector cells that can be both specifically cytotoxic for cells bearing a particular tumor antigen as well as generally tumoricidal.’ The in vivo relevance of macrophage cytotoxicity in the tumor-host relationship remains to be established. A tumor is a complex structure consisting of malignant cells supported by a stroma and a vascular system, both of which are made up of normal cells. In addition, there are also present normal inflammatory cells such as lymphocytes, polymorphonuclear leucocytes and macrophages. It is a serious error to equate “cells in a tumor” with “tumor cells’’; the latter can be in the minority. In this paper we deal with the macrophage content of tumors and its possible relation to the biological properties of the tumor. It must be remembered that macrophages are only one of several nonmalignant cell types that constitute an integral part of a tumor.

Immunologically-mediated restraint of latent tumour metastases.

DIFFERENT grafted sarcomas, all originally from chemically-induced autochthonous tumours, vary widely in their capacity to form metastases, as determined by the incidence of secondaries which appear in the lung and lymph nodes in the months following surgical removal of the ‘primary’ transplant from syngeneic rats1. Tumours which do not form metastases readily in normal animals can be induced to do so by grafting into syngeneic rats depleted of T lymphocytes by procedures which do not compromise the bone marrow1. We concluded that specific immune processes requiring the participation of T lymphocytes contributed to the control of metastatic spread—possibly by reducing the number of tumour cells released because there is an inverse relationship between the number of macrophages within the tumour and metastasis1,2. In addition, circulating tumour cells may be destroyed immunologically and an immunologically-specific factor that acted against intravenously-injected sarcoma cells was demonstrated in the serum and lymph of tumour-bearing rats3,4.

Epidermal growth factor induced tyrosine phosphorylation of nuclear proteins associated with translocation of epidermal growth factor receptor into the nucleus

Treatment of human squamous carcinoma cells (HN5 cells) with epidermal growth factor (EGF) caused a time-dependent increase in tyrosine phosphorylation of six nuclear proteins of molecular mass 166, 140, 117, 95, 86 and 79 kDa. The major tyrosine phosphorylated protein was indistinguishable from the plasma membrane form of the epidermal growth factor receptor and was shown by enzyme linked immunosorbent assay (ELISA) to be translocated into the nucleus from extra-nuclear sites upon ligand stimulation. Using immunoelectron microscopy of both isolated nuclei and whole cells, epidermal growth factor receptor (EGF-R) was found to be associated with the chromatin and, to a lesser extent, with the inner surface of the nuclear membrane. Tyrosine phosphorylation of proteins other than EGF-R was particularly notable in the nucleoli. These observations suggest that EGF-R may exert some of its physiological functions by directly inducing tyrosine phosphorylation of specific nuclear proteins. Translocation of EGF-R to the nucleus may provide a vital link between plasma membrane signalling and gene activation.

Growth inhibitory effects of FK506 and cyclosporin A independent of inhibition of calcineurin

The ability of the immunosuppressive agent FK506 to affect growth of the epidermal growth factor-receptor (EGF-R) overexpressing cell line, A431, was compared with that of the structurally unrelated immunosuppressive compound, cyclosporin A (CyA). Both were shown to inhibit growth, although neither of them caused down-regulation of the EGF-R or affected epidermal growth factor (EGF)-induced tyrosine phosphorylation of the EGF-R. Inhibition of growth was not specific to EGF-R pathways, as both FK506 and CyA also inhibited EGF- and platelet-derived growth factor (PDGF)-induced DNA synthesis in fibroblasts. In all assays FK506 was less potent than CyA even though it is 10-100 times more potent as an immunosuppressive agent. The role of calcineurin in CyA- or FK506-induced growth inhibition was investigated using the synthetic pyrethroid insecticides: cypermethrin, deltamethrin and fenvalerate, which are known calcineurin inhibitors. Failure of these agents to block cell growth or influence growth factor-induced mitogenesis indicated that the biochemical pathway(s) by which CyA or FK506 inhibited cell growth did not depend solely on inhibition of calcineurin.

Contribution of host-derived growth factors to in vivo growth of a transplantable murine mammary carcinoma

The contribution of host-derived growth factors to tumour growth in vivo was studied using the transplantable murine mammary carcinoma, MT1, grown in syngeneic mice. Promotion of growth of the mammary carcinoma by a factor(s) from the host was evident in experiments in which the carcinoma cells were inoculated intraperitoneally. In this environment, tumours develop as multiple solid nodules, each probably arising from an individual cell or a small cluster of cells. Tumour growth was found to occur in the peritoneal cavity following inoculation of 10(3) cells, but an inoculum of as few as ten cells grew if a leucocyte-rich exudate had first been induced. To determine which host-derived growth factors might contribute to growth of MT1, extracts of the tumour were first examined for growth factor activity. Fractionation of tumour extracts by either ion-exchange chromatography or gel filtration revealed several peaks of mitogenic activity, but none of this could be attributed to epidermal growth factor (EGF). Accordingly, an anti-EGF antibody was tested as a putative inhibitor of tumour growth as any effect of this antibody could be ascribed to removal of EGF derived from the host. The antibody was found to have potent anti-tumour activity when tested against MT1 tumours that had been inoculated into the peritoneal cavity. In contrast, the antibody had little effect on growth of the discrete tumour mass which formed when MT1 was transplanted subcutaneously. The results suggest that host-derived EGF contributes to establishment of microcolonies of MT1 carcinoma within the peritoneal cavity. This may be directly, by providing growth factors to supplement those produced by the tumour until it reaches a certain critical mass to sustain autocrine growth, or indirectly, by affecting the production of other growth-stimulatory factors or cytokines.

Studies on the development of human acute myeloid leukaemia xenografts in immune-deprived mice: comparison with cells in short-term culture.

Human AML cells from the blood of a series of patients have been implanted subcutaneously into mice immune-suppressed by thymectomy and total-body irradiation. Solid tumours resulted from 18 out of 19 samples and their growth was compared with the proliferation of AML cells in culture. In 17 cases tumours grew to a maximum size and then spontaneously regressed. Cells from one patient produced tumours which did not regress and could be retransplanted into freshly immune-suppressed mice. Cells from a human promyelocytic cell line (HL60) also produced nonregressing and retransplantantable tumours. Normal human mononuclear bone marrow cells implanted s.c. produced a growth pattern similar to that of the majority of AML cells. A second inoculum of AML cells into animals with regressing tumours also produced tumours and thus regression cannot be accounted for on the basis of returning immunity. AML cells placed into short-term suspension culture invariably matured to monocyte/macrophage type cells and/or granulocytic cells as identified by cytochemical staining. However, no correlation was observed between proliferation or maturation of cells in culture, and tumour growth in vivo. Cells derived from disaggregated AML tumours also showed evidence of myeloid differentiation suggesting that tumour regression is due to maturation of leukaemic cells.

Immunotherapy of human acute leukaemia.

Conclusions are difficult to draw. In the six studies of immunotherapy of AML discussed, all the three employing BCG and cells showed a prolongation of survival and the major contributing factor to this prolongation of survival was extension of life after relapse. In the three studies using BCG alone only one shows a beneficial effect, but some more time must be allowed to elapse before this can be concluded with confidence.

Constrained Peptide Analogues of Transforming Growth Factor-α Residues Cysteine 21-32 Are Mitogenically Active USE OF PROLINE MIMETICS TO ENHANCE BIOLOGICAL POTENCY

Two proline mimetics, the enantiomers of 2-azabicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop β-sheet sequence of human transforming growth factor-α (TGF-α) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 μM; in the presence of low doses of TGF-α, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-α alone. These effects are consistent with the peptides acting via the same mechanism as TGF-α. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the β-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.

Urinary epidermal growth factor (hEGF) levels in patients with carcinomas of the breast, colon and rectum.

A specific two-site ELISA for human epidermal growth factor (hEGF) has been used to measure urinary hEGF/creatinine ratios in 30 normal subjects, 30 hospital in-patients with breast cancer and 30 hospital in-patients with colonic or rectal cancer. There was no significant difference between patients with breast cancer and controls. Although a statistically significant difference between patients with colorectal cancer and controls was observed, the biological significance of this observation is doubtful. No clear effect of the presence of breast or colorectal carcinoma on the urinary excretion of hEGF has been observed.

Concomitant synthesis of growth factors and their receptors ― an aspect of malignant transformation

Sporn and Todaro [1] have suggested that malignant transformation may involve autocrine stimulation by transforming growth factors. In an extension of this hypothesis it is proposed that the malignant phenotype may be characterized by the simultaneous expression of a growth factor and its appropriate receptor, either one of which, but not both can be expressed by normal cells.