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.

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies.

Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.

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.

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.

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.

Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli.

Major capsid proteins (MCPs) of various papillomaviruses have recently been expressed in heterologous cells as soluble and functional polypeptides. The host cells for producing these proteins have so far been of eukaryotic origin; however, E. coli has potential utility a host, with advantages over eukaryotic cells such as relatively simple culture requirements and greater ease of mutation of expressed sequences. We studied the expression by E. coli of the MCP of human papillomavirus type 16 (HPV16) using the gene derived from the 'prototype' HPV16 genome. Using expression vector pTrc99A, the protein was produced in full-length unfused form at levels of 3-4% of cell protein. Soluble polypeptide was detected, albeit at low levels. The level of solubility was not increased by growing cells at low temperature and slowing the rate of protein synthesis. The soluble protein was degraded at its carboxy terminus by an outer membrane protease of E. coli, OmpT, giving rise to two slightly shortened protein species of 52K and 56K in addition to the full-length 57K polypeptide. Since the MCP of prototype HPV16 is known to be prone to excessive aggregation compared with other papillomaviral MCPs, the recovery of soluble polypeptide indicates that E. coli is worth consideration as an alternative host to eukaryotes for producing these proteins.

Human papillomavirus type 16 E6, E7 and L1 and type 18 E7 proteins produced by recombinant baculoviruses.

Proteins derived from the E6, E7 and L1 ORFs of HPV16 and the E7 ORF of HPV18 were produced in insect cells using a baculovirus expression system. HPV ORFs were inserted into baculovirus transfer vectors pAcYM1 or pVL1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. Using HPV-specific antisera and monoclonal antibodies HPV proteins were identified in lysates of Spodoptera frugiperda (Sf-21) cells infected with HPV-recombinant baculovirus. Immunoreactive HPV16 E7 protein produced in Sf-21 cells had an apparent M(r) of 19 kDa, larger than that predicted from the amino acid sequence, and similar to that of native HPV16 E7 protein in HeLa and CaSki cells. The apparent M(r) of recombinant HPV18-E7, HPV16-L1 and HPV16-E6 proteins was equivalent to the M(r) values predicted from the amino acid sequence. Thermostability studies revealed that the half-life of HPV16-E7 protein in Sf-21 cell lysate was approx. 20 h at 4 degrees C, 2 h at 22 degrees C, and less than 30 min at 37 degrees C. HPV16 L1, HPV16 E7 and HPV18 E7 proteins were predominantly localised in the nucleus of recombinant baculovirus-infected Sf-21 cells, whereas recombinant HPV 16 E6 protein was localised in both the cytoplasm and nucleus of infected insect cells. Northern blot analysis of RNA derived from insect cells infected with vAc16E6E7, a recombinant baculovirus containing both HPV16 E6 and E7 ORF's, revealed the presence of only E6 ORF transcripts, suggesting that the splicing of RNA products derived from the E6 and E7 ORF's, as observed in cervical cancer-derived cell lines, is not performed in insect cells. Baculovirus-derived HPV proteins have similar biological properties to the native proteins and should be suitable for studies on the immunology of HPV.