Investigation of the peak action wavelength of light-activated gene transduction.

Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Previous studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, whereas UVA induces LAGT secondary to reactive-oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage and determine an optimal wavelength to maximize safety and efficacy. UV at 288, 311 and 320 nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4 kJ m⁻² at 311 nm. Consistent with its robust cytotoxicity, 288 nm produced significantly high levels of DNA damage at all doses tested, whereas 311, 320 and 330 nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. Although 288 nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ m⁻² at 311 nm. An in vivo pilot study assessing 311 nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (P<0.05) increase in transduction with insignificant cytotoxicity. In conclusion, 311 nm was found to be the optimal wavelength for LAGT on the basis of its superior efficacy at the peak dose and its broad safety range that is remarkably wider than the other UV wavelengths tested.

Decline in MHC class I expression with increasing thickness of cutaneous melanomas in standard-strain transgenic mouse models.

Major histocompatibility complex (MHC) class I proteins are required for the formation of complexes with antigenic peptides that enable cytotoxic T lymphocytes (CTLs) to recognize and lyse target cells. The frequent loss of MHC class I expression reported in human melanomas and melanoma cell lines may therefore be an obstacle to CTL-based immunotherapy. We have investigated the expression of MHC class I proteins in the cutaneous melanomas of Tyr-SV40E (C57BL/6 strain) transgenic mice in order to evaluate their potential as experimental models for immunotherapy. The SV40 large T (LT) oncoprotein, which is expressed exclusively in the melanocytic lineages of these mice, was used as a marker for flow cytometric analysis of the parenchymal (potential target) cells of 35 freshly dissociated samples from 28 primary tumours. All the tumours were ulcerated and exceeded the Breslow thickness indicative of a poor clinical prognosis in human melanoma. Using antibodies against H-2D(b) and H-2K(b) class I proteins, the LT antigen-positive cells were found to have high levels of both these MHC class I molecules in the thinnest tumours (2 mm), whereas the levels tended to decline with increasing tumour thickness. Among the tumours > 4 mm thick, five had no detectable MHC class I expression. Unexpectedly, the apparent loss of H-2D(b) and H-2K(b) proteins was observed not only in LT-positive cells but also in LT-negative cell populations. Expression of both H-2D(b) and H-2K(b) was restored in tumours derived from a class I-low melanoma cell line by treatment of the hosts with interferon-gamma. These results implicate a regulatory defect as a principal cause of the loss of MHC class I antigens, as noted by others in some human tumours, and they demonstrate that this loss is remediable, even in advanced stages of melanomas.

Diagnosis and treatment of carotid artery stenosis.

Diagnosis and management of carotid arterial occlusive disease and its ensuing comorbid illnesses have been the focus of extensive debate during the past two decades. The development of sophisticated ultrasound technology has enabled us to objectively define the severity of carotid stenosis in regions commonly accountable for stroke. Results from several well-designed prospective randomized trials have enhanced our understanding of the epidemiology of this disease and have guided our judgment with regard to aggressive diagnosis and intervention. As clinicians, we have been charged to address underlying risk factors, including hyperlipidemia and smoking abuse, as well as concomitant coronary, renal, and lower extremity artery disease. Carotid endarterectomy coupled with optimal medical management is superior to medical management alone in asymptomatic patients with high-grade carotid stenosis (> 70% diameter reduction) and recently has been shown to have value for symptomatic patients with stenosis of 50% to 69%.

Metastatic cutaneous melanoma promoted by ultraviolet radiation in mice with transgene-initiated low melanoma susceptibility.

An inbred-strain (C57BL/6) transgenic (Tyr-SV40E) mouse model of ultraviolet radiation (UVR)-induced metastatic cutaneous melanoma was produced without the use of chemical carcinogens and without resulting in other skin malignancies. Expression of this transgene occurs specifically in melanocytic-lineage cells. In untreated hemizygous mice of transgenic line 12 there are no skin melanomas, and the oncogenic sequence, which is expressed at a very low level, functions solely as a weak initiating stimulus. UVR [including 65% ultraviolet B (280-320 nm wavelength)] supplied the necessary promoting stimulus leading to melanomas. Of various trial protocols, eight were successful and involved exposure of 112 mice for a limited time on each of 3-10 days starting at 2-3 days of age and totalling 1.1-3.7 J/cm2 UVR. Fourteen of these animals developed a total of 15 invasive skin melanomas on the head and body, arising between 37-115 weeks of age and, therefore, often after a relatively long latency. The tumors were melanotic and in five of the mice they yielded macrometastases in regional and distant sites. The single most favorable protocol (1.9 J/cm2 total UVR, at 0.38 J/cm2/day for 5 days starting at 3 days of age) led to the highest incidence of melanoma (5 of 19 mice) and one of the lowest mortality rates (2 of 19). No melanomas occurred in UVR-treated nontransgenic C57BL/6 controls. Benign skin keratoacanthomas arose and often regressed in treated transgenic as well as nontransgenic mice. This new transgenic mouse model introduces many novel possibilities for experimental analysis of the melanoma-promoting mechanisms of UVR and also of the ability of specific genetic changes to impede or facilitate the UVR effect.

Comparative decreases in tyrosinase, TRP-1, TRP-2, and Pmel 17/silver antigenic proteins from melanotic to amelanotic stages of syngeneic mouse cutaneous melanomas and metastases.

Malignant cutaneous melanomas and metastases were taken directly from in situ lesions of genetically identical (C57BL/6 strain) Tyr-SV40E transgenic mice, and samples were analyzed by Western immunoblotting with antisera specific for the COOH terminus of each of four melanocytic proteins. These were tyrosinase, TRP-1, TRP-2, and Pmel 17/silver. Of the 13 melanomas examined, there were 5 melanotic primary tumors, 5 amelanotic primary tumors, and 3 amelanotic metastases. The melanotic tumors expressed all of the markers to some extent. In contrast, the amelanotic tumors lacked detectable levels of one, two, or three of the proteins, except for an apparently amelanotic tumor sample in which all were expressed, but in which some melanotic cells were likely to have been present. Thus, despite some variability, there is clearly a downward trend in the presence of these proteins as the tumors become amelanotic, a pigmentary change associated with ongoing malignant progression. In the amelanotic tumors, tyrosinase was most often deficient, whereas TRP-2 was most often persistently expressed. These results, obtained from melanomas of syngeneic origin, indicate that tumors in the relatively early stages of malignancy might be more responsive than later-stage tumors to immunotherapy involving an ensemble of antigenic peptides of the tested gene products. Moreover, TRP-2 peptides may be especially useful for therapeutic intervention at the later stages.

Differences in latency and inducibility of mouse skin melanomas depending on the age and anatomic site of the skin.

To determine whether the occurrence of skin melanoma is influenced by the age or the anatomic source of the skin in melanoma-susceptible transgenic mouse models, skin was grafted from donors of different ages or from different anatomic sites to a standard (lateral trunk) site in adult recipients of the same transgenic strain. In 27 grafts of neonatal body skin, melanomas arose with a significantly shorter latency than in 37 grafts of older body skin. The difference may reflect not only the larger number of extrafollicular melanocytes in a given area of neonatal skin but also their unusually high mitotic activity shortly after birth and the influence of other growing skin cells nearby. Each of these body-skin grafts usually developed a single tumor situated near the graft edge. Because maximal wound healing occurs at the edge of such full-thickness skin grafts, melanocytes near the edge would receive the highest exposure to growth factors and cytokines associated with wound healing. In contrast to these results, grafts of snout skin yielded many melanomas, each originating from melanocytes within a vibrissa follicle rather than at the graft edge. The relatively strong local tumorigenic stimulus may be attributable to intrafollicular growth factors normally involved in whisker growth. The above-described experiments support the conclusion that agents in the immediate skin environment of the melanocyte, in addition to the state of the melanocyte itself, contribute to melanoma formation.

The role of open lung biopsy in the management and outcome of patients with diffuse lung disease.

BACKGROUND: Open lung biopsy (OLB) has long been considered the gold standard for the diagnosis of parenchymal lung disease. With recent advances in computed tomographic imaging and diagnostic techniques (eg, bronchoscopy), we thought it necessary to reevaluate the role of OLB in the management of patients with interstitial lung disease. METHODS: We carried out a retrospective analysis of 103 OLBs performed at Hadassah University Hospital, Jerusalem, and Carmel Medical Center, Haifa, between 1980 and 1994. Data gathered included demographic information, underlying condition, indications for biopsy, diagnosis before biopsy, final diagnosis, change in therapy, and mortality. "Benefit" was defined as a change in therapy resulting in survival. RESULTS: There were 45 immunocompetent patients (group 1), 39 immunocompromised patients (group 2), and 26 children (group 3), 7 of whom were included in group 2 for analysis. Overall, a diagnosis was reached after OLB in 85% of patients. An unexpected diagnosis was reached in 52%, and a change in therapy was instituted in 46%. The overall mortality rate was 20%. In group 1, the mortality rate was 13%, and "benefit" from OLB was reached in only 18%. In group 2, the mortality rate was 39%, and "benefit" was achieved in 46%, and in group 3, the mortality rate was 12% and "benefit", 50%. CONCLUSIONS: Open lung biopsy is an excellent diagnostic technique. In immunocompetent patients, the "benefit" is relatively low, as therapy (corticosteroids) is frequently used after biopsy. In immunocompromised patients, therapy changes substantially after OLB, but mortality is high. Therefore, OLB should be reserved for patients in whom the diagnosis is likely to lead to a change in therapy and in patients in whom the underlying condition has a reasonable prognosis according to the clinical impression by the attending physician.

Qualitative and quantitative catalog of tyrosinase alternative transcripts in normal murine skin melanocytes as a basis for detecting melanoma-specific changes.

The decline in cell differentiation commonly associated with malignant progression may be due in part to an increase in alternative splicing of the pre-mRNAs of tissue-specific genes. As a necessary basis for investigating this possibility in a murine model of cutaneous melanoma, the complete qualitative and quantitative inventory of alternative transcripts was sought for the tyrosinase gene in normal mouse skin melanocytes, as this gene plays a key role in melanization. Of 111 alternative mRNAs predicted from known splice sites in the gene, 19 isoforms were detected, and their abundances determined, through a systematized protocol involving splice junction-specific probes, exon-specific restriction enzymes, and quantitative RT-PCR with an RNA internal standard. No unpredicted tyrosinase transcripts were discovered. Two of the transcripts, each involving an intra-exonic deletion and present at relatively low abundance in normal skin, were subsequently found to be consistently upregulated in melanomas.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: utilization in design of syngeneic immunotherapy models.

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase-PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Delta1b and Delta1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably Delta1b) present in C/C were obligatorily absent, others (Delta3 and Delta1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to Delta3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to Delta1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy.

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Base substitution at different alternative splice donor sites of the tyrosinase gene in murine albinism.

The c2j albino mutation at the mouse tyrosinase locus arose spontaneously in the C57BL/6 inbred strain and causes complete absence of melanin synthesis, as does the "classical" c mutation of long-established albino inbred strains. Sequence analysis of c2j cDNA reveals a G-->T point mutation at nt 291, causing an arginine-->leucine substitution in codon 77, where the arginine position has been conserved in vertebrate tyrosinases and tyrosinase-related proteins. While c2j differs from c, in which there is a G-->C mutation at nt 369 causing a cysteine-->serine substitution, both mutations change the G1 position of alternative 5' splice donor sites in exon 1. Both c2j and c abolish the usage of the respective sites for alternative splicing of the tyrosinase pre-mRNA in skin melanocytes. In c2j, there results an almost eightfold increase in activation of the 5' splice site located 78 nt downstream, but in c there is no activation of the intact upstream splice site. Although the tyrosinase mRNA levels are similar in c2j and wildtype, the protein is virtually absent in c2j, as in c, possibly due to proteolytic degradation.

Accelerated growth of melanomas after specific immune destruction of tumor stroma in a mouse model.

Destruction of the entire stroma in a tumor could provide a stringent test of the prospects for tumor eradication in a single treatment. This possibility was investigated by experimental immune destruction of the stroma in a mouse melanoma model. Melanomas were first produced by grafting skin from transgenic C57BL/6 females of high-melanoma susceptibility to low-susceptibility transgenic males so that the malignant cells would be genetically female and the stromal cells genetically male. Subcutaneous transplant lines were then established from the melanotic and the amelanotic zones of such a melanoma and were carried in transgenic male hosts to ensure the male composition of the stroma. Thus, the male-specific H-Y histocompatibility antigen, which is ubiquitously expressed on male cells, could provide the target for an immune attack against the stroma. The transplant lines were next passaged once in transgenic females preimmunized against the H-Y antigen by having received and rejected a graft of C57BL/6 nontransgenic male skin. The antistromal immune response of these hosts did not prevent recovery of the tumors, which required a substantially prolonged latency. However, after retransplantation to nonimmunized males and females, the latency was markedly shortened from the original level. Thus, the treatment had indirectly selected for more rapidly growing tumor cells and hastened malignant progression.

Expression of plasminogen activators and plasminogen activator inhibitors in cutaneous melanomas of transgenic melanoma-susceptible mice.

The Tyr-SV40E transgenic mouse model of malignant skin melanoma has been used here to generate melanomas in genetically identical (C57BL/6) mice for analysis of the plasminogen activator (PA) system during tumor development and progression. Twenty-two melanocytic lesions were examined by in situ zymography for PA activity and by immunohistochemistry for concomitant visualization of PA proteins; these lesions encompassed 3 nevi and 19 primary melanomas ranging from melanotic through mixed tumors to amelanotic tumors. Although urokinase-type plasminogen activator (u-Pa) activity was not detected at premalignant stages, it began to appear early in tumorigenesis and became more prominent in later stages of a majority of the tumors. The activity was largely attributable to the endothelium of sprouting capillaries and to a lesser degree to granulocytes, fibroblastic cells, and occasional melanoma cells within tumors. Tissue-type plasminogen activator (t-PA) was undetectable or low in all cases. Of the inhibitors (PAI), PAI-1 was seen in endothelial and fibroblastic cells and in the extracellular matrix, whereas PAI-2 occurred in only one case and was melanoma cell associated. Eleven additional melanomas were analyzed by reverse transcription-PCR for PA expression in RNA extracts from relatively large tumor samples. These were obtained from eight primary melanomas and three metastases, again spanning melanotic, mixed, and amelanotic cases. From four of the mixed primary tumors with distinct melanotic and amelanotic zones, the respective components were propagated separately in transgenic hosts as s.c. transplants to obtain data for clearly identifiable melanotic versus amelanotic parts. u-PA and PAI-1 mRNAs were expressed in all. t-PA expression varied greatly and was notably high in several amelanotic tumors or tumor components, possibly as a result of large blood vessels, as such vessels were seen to be t-PA positive in normal tissue. The u-PA activity in sprouting capillaries may indicate a role in neoangiogenesis. Therefore, according to these mouse models, u-PA may indirectly be a potential therapeutic target against melanoma progression.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.

Ultraviolet radiation-induced malignant skin melanoma in melanoma-susceptible transgenic mice.

A new mouse model of UV radiation-induced melanoma is described. Unlike previous models, melanoma induction requires only short-term irradiation, does not require the application of chemical carcinogens, and does not cause any other tumors. The model takes advantage of the fact that Tyr-SV40E (C57BL/6 strain) transgenic mice are all melanoma-susceptible, and that different inbred lines are susceptible to different extents. Four-day-old mice of a moderately susceptible line (line 9 transgenic homozygotes) were exposed for 20 min/day to 328 mJ/cm2 to UVB (280-320 nm wavelength, comprising 70% of the total irradiance) for up to 4 consecutive days. Melanocytic lesions resembling macules, nevi, or early melanomas gradually appeared in the irradiated mice that were not seen in unirradiated transgenic controls of similar age. To afford ongoing observation beyond the short life span of line 9 homozygous mice, skin samples containing a total of 26 selected lesions were grafted at 20 weeks after UV radiation to longer-lived unirradiated hosts of transgenic line 12, in which melanoma susceptibility is low. Ten lesions in the grafts became melanomas; all melanomas had ulcerated and two had metastasized. At the stages examined, all the tumors were deeply melanotic. The remaining 16 lesions were still indolent when the experiment was terminated at 57 weeks post-UV radiation. The present protocol lends itself to variations in the choice of transgenic line, the age of the treated mice, and the intensity and duration of ultraviolet light; appropriate combinations of these variables would be expected to yield melanomas in relatively long-lived transgenic mice without skin grafting. The new model provides an opportunity to determine the melanoma action spectrum, to characterize at the molecular level the melanomas induced by ultraviolet light in comparison with those of other origins, and to investigate in vivo the photoprotective role of melanin.

Transgenic mouse model of malignant skin melanoma.

Tyr-SV40E transgenic mice are specifically susceptible to melanoma due to expression of the oncogene in pigment cells. Mice of the more susceptible lines die young of early-onset eye melanomas, when skin melanomas are still infrequent and benign. To surmount this obstacle, skin from donors of two high-susceptibility lines was grafted to Tyr-SV40E hosts of a low-susceptibility line of the same inbred strain, thereby enabling the skin to outlive the donors and continue to grow in immunocompetent but tolerant hosts. Unexpectedly, donor pigment cells in all the grafts soon selectively proliferated close to areas of greatest wound healing, forming a dense black tracery, especially at the outer rim of the grafts. These lesions slowly grew radially within the grafts, producing irregular greyish patches. Local vertical thickenings then appeared and developed into small melanomas, which soon ulcerated through the epidermis. The tumors rapidly enlarged and became deeply invasive. Discrete black nevi also arose, with many becoming larger and distinctly blue, but those not near areas of pronounced wound healing did not progress to malignancy. In this first series, malignant melanoma resulted in all the grafts from the more susceptible of two donor lines and in some grafts from the other line. Distant metastases occurred in some cases from each line. Most tumors were hypomelanotic and heterogeneous, with lobes or areas differing in melanization. The results strongly suggest that growth factors and cytokines--known to be produced in wound repair--are triggering the growth and malignant conversion of these genetically susceptible melanocytes and that in the graft situation we are merely witnessing a caricature--a usefully exaggerated manifestation of the true events underlying the genesis of melanomas. The striking resemblance to the human malignancy, the genetic uniformity and different susceptibilities of the transgenic lines, and the experimental possibilities in the grafted mice all make them an excellent model of the disease.

Histopathogenesis of malignant skin melanoma induced in genetically susceptible transgenic mice.

Animal models of human malignant skin melanoma were created in melanoma-susceptible inbred-strain transgenic mice by grafting skin from donors of high-susceptibility lines to hosts of a low-susceptibility line, thereby overcoming the problem of early death of the more susceptible animals from eye melanomas. As already described [Mintz, B. & Silvers, W. K. (1993) Proc. Natl. Acad. Sci. USA 90, 8817-8821], melanocytes within the grafts selectively proliferated in close proximity to areas of greatest wound healing, presumably in response to mitogenic factors from cells contributing to wound repair. An orderly sequence of externally visible events culminated in malignant melanoma. We examine here the histogenetic concomitants of these changes and find that they define a stepwise sequence strikingly comparable to that leading to human cutaneous melanoma. Moreover, the histological details suggest some of the underlying mechanisms. While the early lesions are first seen in the superficial dermis in the mouse, and in the basal layer of the epidermis in the human, both progress by radial growth followed by vertical growth. Melanocytic hyperplasia resulted in nests of densely melanized fusiform cells which were losing their dendrites. Some discrete lesions in the deep dermis appeared as blue nevi. As radial proliferation advanced, cellular atypia increased and the previously independent melanocytes cohered closely and formed a small solid tumor; the cells were usually then hypomelanotic or amelanotic. Ulceration of tumor through the epidermis occurred early. The tumor mass grew rapidly in the deep dermis and invaded and destroyed subcutaneous tissue and muscle. Primary tumors in the skin were often heterogeneous, with lobules or regions differing in pigmentation or atypia. However, the cells in circulating emboli, or in metastases in lymph nodes and lungs, appeared relatively homogeneous. These genetically uniform transgenic mouse models provide experimental access to the multistage genesis of melanoma.

Melanocyte culture lines from Tyr-SV40E transgenic mice: models for the molecular genetic evolution of malignant melanoma.

Transgenic Tyr-SV40E mice previously produced on the C57BL/6 inbred-strain background, with SV40 oncogenic sequences specifically expressed in pigment cells, are predisposed to melanoma [Bradl, M., Klein-Szanto, A., Porter, S. & Mintz, B. (1991). Proc. Natl. Acad. Sci. USA, 88, 164-168]. Separate lines of these animals differ genetically only in the number of copies and chromosomal site of integration of the transgene. Skin melanocytes from young mice with no apparent skin lesions were established in continuous culture from hemizygous donors with low, medium and high numbers of transgene copies, and from a homozygous offspring of the low-copy mouse line. The standard culture conditions enable C57BL/6 wild-type melanocytes to become stably immortalized without transformation. The transgenic cell lines all changed over time in an orderly progression. However, with greater numbers of transgene copies, the cells more rapidly displayed shorter doubling times, increased anchorage independence, reduced serum and growth factor requirements, decreased tyrosinase expression and melanin content, increased oncogene expression, and capacity to form malignant melanomas when tested by grafting. Melanocytes with the lowest number of transgene copies were of special interest. They grew more rapidly than the wild-type cells from the outset, but did not become tumorigenic until an apparently small number of still-unknown genetic changes had spontaneously occurred, or until the number of transgene copies was increased slightly by homozygosity. In contrast to the hemizygous low-copy cells, the homozygous counterparts underwent striking and rapid transformational changes and early conversion to malignancy. Thus such low-copy transgenic melanocyte lines afford an exceptional opportunity for molecular analysis of somatic genetic evolution toward malignant melanoma.