Bis-naphthalimides 3: synthesis and antitumor activity of N,N'-bis[2-(1,8-naphthalimido)-ethyl] alkanediamines.

The bis-naphthalimides are a new class of antitumor agent. Previously, we have described initial synthetic series which established that two naphthalimide chromophores joined by polyamine linkers can produce agents with antitumor activity. We now extend these studies to describe the synthesis of a N,N'-bis[1,8-naphthalimido)-ethyl]alkanediamine series and examine the alkyl linker and chromophore substitution requirements for optimal antitumor activity. As a result, a bis-naphthal-imidoethyl derivative with no chromophore substitution and a NH-(CH2)3-NH bridge was identified. This agent (LU 79553) had both potent cellular cytotoxicity (IC50 = 0.014 microM) and was curative against MX-1 tumors grown in athymic mice. It has now been selected for clinical development.

Involvement of integrin alpha V gene expression in human melanoma tumorigenicity.

Human melanoma originates in the skin and can lead to wide-spread metastatic disease. Analysis of melanoma biopsy material has shown that the vitronectin receptor, integrin alpha v beta 3, is a specific marker of the most malignant cells, i.e., vertically invasive primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymic nude mice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo.

Suppression of spontaneous melanoma metastasis in scid mice with an antibody to the epidermal growth factor receptor.

The human melanoma cell line M24met metastasizes spontaneously from s.c. tumors to multiple distant sites in mice with severe combined immunodeficiency. Metastasis to lymph nodes and lungs is found in 100% of the animals. M24met has an undifferentiated phenotype and extra copies of the short arm of chromosome 7. This cell line expresses the epidermal growth factor receptor, and 425.3, a monoclonal antibody to the epidermal growth factor receptor, binds to 291,000 receptor molecules per M24met cell with a KD of 2.3 x 10(-10) M. This antibody has no effect on the proliferation of M24met cells under tissue culture conditions and does not mediate effector cell or complement-dependent cytotoxicity of these cells in vitro. However, treatment of established s.c. M24met tumors in mice with severe combined immunodeficiency with monoclonal antibody 425.3 specifically suppresses spontaneous metastasis of these tumors. Total doses of 4, 2, and 1 mg antibody per mouse decrease the number and size of melanoma metastases and prolong the life span of treated animals. Treatment with 4 mg of the F(ab')2 fragment of monoclonal antibody 425.3 does not influence M24met melanoma metastasis, implying a significant contribution of the Fc portion to the antimetastatic effect of this antibody.

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody.

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.

Specificity of antigens on UV radiation-induced antigenic tumor cell variants measured in vitro and in vivo.

In vitro exposure of nonimmunogenic murine tumor cells to UV radiation (UVR) generates highly antigenic variants that are immunologically rejected by normal, syngeneic mice. The purpose of this study was to determine whether these antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants. Furthermore, upon transplantation, the UVR-induced antigenic variants grew in UV-irradiated and immunosuppressed mice, but not in untreated mice indicating that the variants expressed the determinant recognized by suppressor T-cells present in UV-irradiated mice. These results demonstrate that highly antigenic cells generated by the in vitro exposure of two different murine tumors to UV radiation express a determinant shared with the parental tumor cells and nonimmunogenic clones, a unique variant-specific determinant and the suppressor cell-defined determinant present on UVR-induced tumors. Based on these results, two models are proposed to explain the make-up of the antigenic determinants present on the UVR-induced antigenic variants.

The role of ultraviolet radiation in the induction of melanocytic skin tumors in inbred mice.

The purpose of these studies was to determine whether ultraviolet radiation contributes to the induction of cutaneous melanocytic tumors in mice. At 4 days of age, C3H/HeNCr(MTV-) mice received an initiating dose of 7,12-dimethylbenz(a)anthracene, followed by twice weekly applications of croton oil. Of the mice treated with this protocol, 31% developed melanoma. The addition of chronic doses of ultraviolet radiation (UVR) to the carcinogen treated site dramatically accelerated the development of the melanomas. Substituting UVR for the initiator or the promoter in this two-stage carcinogenesis model resulted in the development of a few melanocytic tumors (less than 10%). These experiments demonstrate that UVR accelerated the development of cutaneous melanocytic tumors induced in mice by chemical carcinogens and participated in the induction of these tumors by serving as a weak initiator or promoter.

Effect of ultraviolet-B radiation on the in vivo growth of murine melanoma cells.

The role of UV radiation in the development of malignant melanoma has yet to be clearly defined. The purpose of these studies was to determine whether UV irradiation of mice produces local or systemic alterations that increase the in vivo growth of transplanted melanoma cells. K-1735 melanoma cells were injected into the external ears of syngeneic C3H mice. UV irradiation of the mice before or at the time of injection of the melanoma cells accelerated the appearance of the tumors. The effect was observed when melanoma cells were transplanted directly into the site of UV irradiation, but not when they were injected into an unirradiated site. The initial survival of radiolabeled melanoma cells at the site of inoculation was not altered by UV irradiation of the host, suggesting that the accelerated appearance of tumors was due to an increase in the clonogenic potential of cells injected into UV-irradiated skin. The effect of UV irradiation on the development of other syngeneic tumors was also investigated. The outgrowth of a second melanoma was also accelerated in UV-irradiated mice, whereas the growth of a UV-induced fibrosarcoma, a methylcholanthrene-induced fibrosarcoma, and a spontaneous hepatocarcinoma was not affected. These results suggest that, in addition to its carcinogenic activity, UV radiation may contribute to the incidence of cutaneous melanoma because of a local effect on the skin that stimulates melanoma development.

Role of helper T-lymphocytes in rejection of UV-induced murine skin cancers.

The purpose of this study was to examine the role of helper T-lymphocytes (Th) in the immunological rejection of UV-induced tumors. Mice repeatedly exposed to UV radiation develop suppressor T-lymphocytes that facilitate the growth of UV-induced tumors by interfering with host immunity. These suppressor cells specifically blocked the generation of antitumor Th, suggesting that Th may be important in the immunological rejection of UV-induced tumors. The Th activity generated by a UV-induced tumor that grows progressively in normal mice was compared with that generated by a highly antigenic, regressor clone of the same tumor. The regressing tumor cell line generated a much higher amount of Th activity than the parental, progressor tumor cell line. The amount of Th activity generated by a highly antigenic, UV-induced tumor injected into normal mice, in which it regresses, was compared to the Th activity in UV-irradiated mice, in which the tumor grows progressively. Again, tumor regression was associated with a higher amount of Th activity, and this increased activity was detectable in the environment of the regressing tumor. Lyt-1+ cells containing Th activity mediated the regression of a UV-induced tumor when injected with the tumor cells s.c. into immunosuppressed mice. Lyt-1- cells were cytotoxic to tumor cells in vitro but were unable to cause tumor rejection in vivo. These studies suggest that Th play a central role in the immunological rejection of UV-induced tumors.

Regulation of the immune response against UV-induced skin cancers: specificity of helper cells and their susceptibility to UV-induced suppressor cells.

The purpose of this study was to determine the role of T helper (Th) cells in the immune response to UV-induced tumors. Repeated exposure of mice to UV radiation results in the production of suppressor T lymphocytes that facilitate tumor growth by inhibiting host immunity. To investigate whether the suppressor T cells inhibit the response to UV tumors by blocking the generation of Th, we employed an indirect method for measuring helper cell activity. We found that Th were produced in normal mice after immunization with UV-induced tumors. These Th appeared to be specific for the immunizing tumors, in contrast to the UV-induced suppressor cells, which recognize UV-induced tumors as a group. The suppressor T cells responsible for inhibiting tumor rejection have no effect on tumor-specific helper cell activity in vitro. However, UV-induced suppressor T cells transferred into unirradiated mice seem to block the generation of helper cell activity after immunization with UV-produced tumors. These results suggest the UV-induced suppressor cells may prevent tumor rejection by blocking the generation of Th.

Advances in the immunobiology of the skin. Implications for cutaneous malignancies.

Recent advances in cutaneous immunobiology have led to the realization that skin is an important and unique immunologic organ. Studies on the immunobiology of skin cancers induced by ultraviolet radiation indicate that immune mechanisms can play a crucial role in the development of cutaneous tumors. This paper summarizes the evidence linking skin and the immune system and discusses current hypotheses concerning the mechanisms by which UV radiation interferes with cutaneous immunity. The significance of these findings for cutaneous carcinogenesis is discussed.

Variation in agar growth of transformed 3T3 cells after tumor formation in nude mice.

Cells from a cloned line of spontaneously transformed 3T3 cells had a colony-forming efficiency in agar (CFEag) of about 10-15% and induced poorly differentiated sarcomas when injected into nude mice. These tumor cells were recultured in vitro and tested for their ability to grow in suspension. Initially, the tumor cells had a CFEag which was a hundredfold to a thousandfold lower than the cells that had been grown only in vitro. After 5-8 further weekly passages, however, the tumor lines recovered their original ability to grow in agar. For determination as to whether this increased CFEag was due to selection of cells with a higher CFEag from the tumor cell population or to adaptation of many of the tumor cells to agar growth, clones were isolated directly from a primary tumor, and each was tested weekly for agar colony formation. All of the tumor clones, as well as the uncloned tumor line, were able to recover their original ability to grow in agar. However, one tumor clone had a relatively high CFEag in the first assay, so the selection hypothesis could not be totally excluded. The initial low CFEag of the recultured cells was not due to the presence of normal nude mouse cells in the population. Before in vivo growth no clones could be isolated from the sublines that had as low a CFEag as the tumor cells isolated after in vivo growth. Tumor cells that had been repeatedly passaged in vivo still had a much lower CFEag than the input cells upon explantation into culture. The results suggest that phenotypic alterations observed during tumor growth and subsequent cultivation have an epigenetic basis.

Variation in capacity for anchorage-independent growth among agar-derived clones of spontaneously transformed BALB/3T3 cells.

A subline of cloned spontaneously transformed BALB/3T3 cells had a colony-forming efficiency (CFE) in agar of 5 to 20%. Individual agar colonies isolated and reseeded into agar were not significantly more efficient at initiating colonies than the original unselected subline. Four successive cycles of agar growth and selection also failed to increase the mean CFE in agar. Randomly selected clones isolated on a plastic surface all had the capacity to grow in agar. These results suggest that the failure of the majority of the cells to grow in agar is not the result of an intrinsic or heritable inability to do so. The ability to initiate a colony in agar seems to vary phenotypically from cell to cell. In contrast, agar colonies isolated from some tumor cell lines (originating from related spontaneously transformed 3T3 cells) and reseeded in agar had a higher CFE than the unselected tumor cell lines. In one case, this increased CFE in agar was lost when the cells were passaged on plastic without further selection for agar growth. Thus, expression of the anchorage-independent phenotype may vary, even among related cloned populations of transformed cells.

Colony morphology and heritability of anchorage-independent growth among spontaneously transformed Balb/3T3 cells.

A clone of spontaneously transformed Balb/3T3 cells produced four different types of colonies in agar. Flat colonies arose at the interface between bottom and top layers of agar, two types of somewhat flattened compact colonies arose within the top layer, and mixed colonies developed when the compact colonies contacted the interface. The proportions of different colony types could be altered by varying the volume, concentration, and gelling rate of the agar. Spherical colonies only appeared in ungelled medium. None of the colony types bred true, showing that their morphologies depended on local conditions. When cells from a subpopulation with low colony-forming efficiency in agar (CFE) were cloned on plastic, all clones formed colonies in agar and did so with about the same CFE as the parental population. Colonies isolated from agar had no higher CFE than the parental population. The results indicate a great phenotypic heterogeneity in anchorage-independent growth, even in cloned populations of transformed cells.