The expression of Ca 2ϩ channel A1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned A1E cDNA sequences led to the prediction of A1E splice variants. Transcripts of two cloned A1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter A1E isoform have been assigned to the rat cerebrum and to neuron-like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)-PCR and visualized on the protein level by Western blotting with common and isoformspecific antibodies. Transcripts of the longer A1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS-1 (rat) and βTC-3 (mouse), in the pituitary cell line AtT-20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of A1E in cell lines and tissues shows that the wide expression of A1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called A1Ee has been determined by RT-PCR, while in cerebellum a set of different A1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue-specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform A1Ee in islets of Langerhans and kidney leads to the suggestion that A1E might be involved in the modulation of the Ca 2ϩ -mediated hormone secretion.Keywords : A1E isoforms ; Ca 2ϩ channels ; islets of Langerhans; kidney; single-cell RT-PCR.Low and high voltage-gated Ca 2ϩ channels are subdivided, according to their threshold of activation and their different single channel conductances [1]. The pore-forming subunit of the first low voltage-activated T-type Ca 2ϩ channel has been cloned and functionally expressed recently as A1G [2].The group of six different high voltage-activated Ca 2ϩ channels (A1S-, A1C-, A1D-, A1A-, A1B-, A1E subunits) has been investigated intensively during the last decade [3,4]. They are subdivided into two different families, based on their primary sequence. The A1S, A1C, and A1D subunits are sensitive against the classical blockers of L-type Ca 2ϩ channels, the dihydropyridines, the phenylalkylamines and the benzothiazepines. The subfamily of the non-L-type A1 subunits share, among each other, a functional interaction with inhibitory G proteins [5]. The Ntype Ca 2ϩ channels (A1B) are blocked by ω-conotoxin-GVIA, the P-type and Q-type Ca 2ϩ channels (A1A) are discriminated byCorrespondence to T. Schneider,

BACKGROUND: Individuals with Fanconi anemia (FA) have a 500-fold to 700-fold elevated risk, much earlier onset, and limited therapeutic options for oral squamous cell carcinoma (SCC) compared with the general population. The early detection of SCC, or preferably its precursors, is mandatory to retain curative therapeutic options. Due to frequent synchronic and metachronic oral lesions, tissue biopsies, as usually recommended by guidelines, often are not feasible. In the current study, an alternative strategy for early detection using oral brush biopsy-based cytology was validated regarding its diagnostic accuracy. METHODS: Over a 12-year period, the oral cavities of a large cohort of 713 individuals with FA were inspected systematically and brush biopsy-based cytology of 1233 visible oral lesions was performed. In cases of inconclusive cytology, analysis of DNA ploidy was performed whenever possible. The results were correlated to a long-term clinicopathological follow-up reference standard. RESULTS: A total of 737 lesions were suitable for statistical analysis, including 86 lesions with at least high-grade oral epithelial dysplasia in 30 patients. For cytology, the sensitivity and specificity were 97.7% and 84.5%, respectively. Additional analysis of DNA ploidy increased the sensitivity and specificity to 100% and 92.2%, respectively. CONCLUSIONS: Careful inspection of the oral cavity of individuals with FA followed by brush biopsy-based cytology appears to identify visible oral, potentially malignant and malignant lesions that warrant treatment. Approximately 63% of SCC and precursor lesions are detected at a noninvasive or early stage. Negative cytology or a lack of DNA aneuploidy can exclude high-grade oral epithelial dysplasia or SCC with high accuracy and thus reduce the need for invasive diagnostic biopsies. Cancer Cytopathol 2020;128:403-413.Attri butio n-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

S U M M A R YThe calcium channel ␣ 1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of ␣ 1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac ␣ 1E was determined by SDS-PAGE and immunoblotting (218 Ϯ 6 kD; n ϭ 3). Compared to ␣ 1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of ␣ 1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of ␣ 1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of ␣ 1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of ␣ 1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca 2 ϩ -dependent secretion of peptide hormones such as ANP and urodilatin.

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SUMMARY Polyclonal antibodies were raised against a common and a specific epitope present only in longer ␣ 1E isoforms of voltage-gated Ca 2 ϩ channels, yielding an "anti-Ecom" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an ␣ 1E splice variant with a longer carboxy terminus, the so-called ␣ 1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter ␣ 1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer ␣ 1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, ␣ 1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. ␣ 1E therefore appears to be a common voltagegated Ca 2 ϩ channel linked to neuroendocrine and related systems of the body.