Structure-activity studies of antitumor agent irofulven (hydroxymethylacylfulvene) and analogues.

Many analogues of the antitumor agent irofulven have been readily prepared by replacing the allylic hydroxyl with a variety of nucleophiles. Analogues of acylfulvene (the precursor to irofulven) were also prepared by Michael reaction with acrolein. The toxicity of the analogues was determined, as well as preclinical antitumor activity. Several analogues exhibited good activity in mouse xenografts. Structural requirements for activity are discussed.

Efficacy of MGI 114 (HMAF) against the MRP+ metastatic MV522 lung carcinoma xenograft.

This study is part of an effort to evaluate efficacy of the novel agent MGI 114 (HMAF) against tumors resistant to conventional chemotherapeutic agents. MGI 114 is a novel semisynthetic anticancer agent currently in chemotherapeutic phase II trials to evaluate activity against various solid tumors. Previous studies indicate MGI 114 was active against human MDR1/gp170+ solid tumor xenografts. Recent evidence suggests overexpression of the MRP protein may also be clinically relevant to development of drug resistance in solid tumors. We evaluated the efficacy of MGI 114 against a human MRP+ lung carcinoma xenograft. Parent MV522 lung carcinoma cells were transfected with a MRP cDNA expression vector and resistant cells selected by exposure to vinblastine (30-fold resistance). Analysis of resistant clones indicated 20- to 40-fold increases in expression of both MRP mRNA and MRP protein. Administration of MGI 114 at the maximum tolerated dose (7 mg/kg, 5 x/week for 3 weeks) to MRP tumor-bearing mice demonstrated this novel agent was active against MRP+ tumors and significantly extended their lifespan (p<0.001). In contrast, other cytotoxic agents had minimal activity against this MRP+ xenograft. These results indicate MGI 114 should retain activity in vivo against MRP+ tumor types. The development of this MRP+ xenograft model, in conjunction with the parent MV522 and MDR1/gp170+ xenograft models, will be useful for screening new classes of agents for activity against multidrug-resistant tumors.

Structural organization of the microsomal glutathione S-transferase gene (MGST1) on chromosome 12p13.1-13.2. Identification of the correct promoter region and demonstration of transcriptional regulation in response to oxidative stress.

The structure and regulation of the microsomal glutathione S-transferase gene (MGST1) are considerably more complex than originally perceived to be. The MGST1 gene has two alternative first exons and is located in the 12p13.1-13.2 region. Two other potential first exons were determined to be nonfunctional. The region between the functional first exons cannot direct transcription. Thus, one common promoter element directing transcription exists, and RNA splicing occurs such that only one of the first exons (containing only untranslated mRNA) is incorporated into each mRNA species with common downstream exons. MGST1 expression and regulation are therefore similar to those of other hepatic xenobiotic handling enzymes, which also produce mRNA species differing only in the 5'-untranslated regions to yield identical proteins. MGST1 was previously considered a "housekeeping" gene, as non-oxidant inducers had little effect on activity. However, the promoter region immediately upstream of the dominant first exon transcriptionally responds to oxidative stress. In this respect, MGST1 is similar to glutathione peroxidases that also transcriptionally respond to oxidative stress. The discovery that MGST1 utilizes alternative first exon splicing eliminates a problem with the first description of MGST1 cDNA in that it appeared that MGST1 expression was in violation of the ribosomal scanning model. The identification that the first exon originally noted is in fact a minor alternative first exon far downstream of the primary first exon eliminates this conundrum.

(Hydroxymethyl)acylfulvene: an illudin derivative with superior antitumor properties.

Reaction of the fungal sesquiterpene illudin S with excess paraformaldehyde in dilute H2SO4 gives (hydroxymethyl)acylfulvene. The primary allylic hydroxyl thus formed can undergo very facile replacement by a variety of nucleophiles. (Hydroxymethyl)acylfulvene (MGI.114) was more toxic than a precursor, acylfulvene, but less toxic than the parent compound illudin S to HL 60 cells.

Acylfulvenes, a new class of potent antitumor agents.

Acylfulvene, derived from the sesquiterpene illudin S by treatment with acid (reverse Prins reaction), is far less reactive to thiols than illudin S. However, it is reduced readily to an aromatic product, in the same way as illudin S. This may explain its greatly improved therapeutic index compared to that of the parent compound.

Cloned low metastatic variants from human lung carcinoma metastases.

Clonal subpopulations of neoplastic cells were derived, in soft agar, from the spontaneously metastazing variant (MV522) of a human lung carcinoma cell line. The ability of these clones to spontaneously metastasize from subcutaneous sites in athymic mice was then tested. A variation in metastatic ability was expected with the derivation of some low metastatic clones and some high metastatic ones. However, all of the derived clones, although equally tumorigenic, were less metastatic than the parental variant. These clonal cell lines can now be used in a systematic analysis of events associated with the reversion to a less malignant state.

Spontaneously metastasizing variants of a human lung carcinoma cell line: monoclonal antibody characterization.

Spontaneously occurring microscopic lung and lymph node metastases (in athymic mice) of a low metastatic human lung carcinoma cell line, UCP3, and its high metastatic variant, MV522, were isolated. Characterization of the variants included karyotypic and isoenzyme analyses; assessment of spontaneous metastatic capabilities in athymic mice, and monoclonal antibody analyses. The high metastatic variant LNT had barely detectable amounts of a glycoprotein molecule with apparent Mr 73 kd and 90 kd, which was present in the other cell lines. This molecule was detected in 20/24 primary human neoplasms but only in 3/18 metastatic neoplasms, suggesting a loss during the metastatic disease process.