Purpose: The epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) has shown antitumor activity in clinical trials against cancers, such as non^small cell lung cancer and head and neck squamous cell carcinoma (HNSCC). Research on non^small cell lung cancer has elucidated factors that may predict response to gefitinib. Less is known about molecular markers that may predict response to gefitinib in HNSCC patients. Experimental Design: We analyzed possible associations of responsiveness to gefitinib with molecular markers of the EGFR/ErbB receptor family signaling pathway using 10 established HNSCC lines in vitro. IC 50 of gefitinib sensitivity was determined using clonogenic survival assays. ErbB signaling was assessed byWestern and real-time reverse transcription-PCR analyses of EGFR, ErbB2, ErbB3, and ErbB4 expression levels as well as by phosphorylation analysis of pEGFR, pErbB2, pErbB3, pAkt, and pErk. EGFR sequences encoding kinase domain and EGFR gene copy numbers were determined by cDNA sequencing and real-time PCR, respectively. Finally, responsiveness to gefitinib was compared with responsiveness to the anti-EGFR antibody cetuximab (Erbitux). Results: Expression levels of pErbB2 (P = 0.02) and total ErbB3 protein (P = 0.02) associated with resistance to gefitinib. Combining gefitinib with pertuzumab (Omnitarg), an antibody targeting ErbB2 heterodimerization, provided additional growth-inhibitory effect over gefitinib alone on relatively gefitinib-resistant HNSCC cell lines. The same markers did not predict resistance to cetuximab. In contrast, a similar trend suggesting association between EGFR gene copy number and drug sensitivity was observed for both gefitinib (P = 0.0498) and cetuximab (P = 0.053). No activating EGFR mutations were identified. Conclusions: EGFR amplification may predict sensitivity to gefitinib in HNSCC. However, other EGFR/ErbB receptor family members than EGFR may contribute to resistance to gefitinib. ErbB2 and ErbB3 may have potential as predictive markers and as therapeutic targets for combination therapy in treatment of HNSCC with gefitinib.

The expression of mRNAs for a transition protein (TP1) and two variants of protamines (P1 and P2) during rat and mouse spermiogenesis was investigated using cDNA hybridization techniques. Slot-blot analyses from 1-mm segments of seminiferous tubules and in situ hybridization from testis sections showed that the levels of mRNA for TP1 increased in step-7 round spermatids at substage VIIb of the seminiferous epithelial cycle, earlier than that of P1 and P2 at substage VIIc. The mRNA levels of all transcripts remained high during steps 8-13 in both species. In the rat, the mRNA of TP1 disappeared during step 14 between substages XIVa and XIVb. The P1 mRNA levels decreased during steps 15-16 (stages I-III) and the P2 mRNA during step 15 (stage I). In the mouse, TP1 mRNA disappeared during step 13 (stage I). The P1 mRNA level decreased before P2 in step 14 (stage II), whereas P2 was detected up to step 15 (stage V). Northern-blot analyses with all three cDNA probes revealed two sizes of mRNA and their stage-specific expression. The shorter transcripts appeared later than the longer ones, at the steps of spermiogenesis where translation is known to begin. The results suggest that transcription of TP1, P1, and P2 mRNAs starts at specifically defined times during spermiogenesis and that the temporal translational regulation of these mRNAs is different.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 alpha, and labeled with [3H]thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiated DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 alpha stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 alpha seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.

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